2&#39;-Modified Oligonucleotides

ABSTRACT

Compositions and methods are provided for the treatment and diagnosis of diseases amenable to modulation of the production of selected proteins. In accordance with preferred embodiments, oligonucleotides and oligonucleotide analogs are provided which are specifically hybridizable with a selected sequence of RNA or DNA wherein at least one of the 2′-deoxyfuranosyl moieties of the nucleoside unit is modified. Treatment of diseases caused by various viruses and other causative agents is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/352,586, filed Jan. 28, 2007; which is a continuation of U.S. patentapplication Ser. No. 09/389,283, filed Sep. 2, 1999, now issued as U.S.Pat. No. 6,531,584; which is a divisional of U.S. patent applicationSer. No. 09/035,357, filed Mar. 5, 1998, now issued as U.S. Pat. No.6,005,087; which is a continuation of U.S. patent application Ser. No.08/468,037, filed Jun. 6, 1995, now issued as U.S. Pat. No. 5,859,221;which is a continuation-in-part of U.S. patent application Ser. No.07/835,932, filed Mar. 5, 1992, now issued as U.S. Pat. No. 5,670,633;which is a U.S. National Phase Application of PCT/US91/05720, filed onAug. 12, 1991; which is a continuation-in-part PCT application of U.S.patent application Ser. No. 07/566,977, filed Aug. 13, 1990, nowabandoned. Each of the above-mentioned applications is commonly assignedwith this application, and the entire disclosures of each are hereinincorporated by reference.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledISIS5137USC1SEQ.TXT, created on Jun. 15, 2007 which is 8 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention is directed to nuclease resistant oligonucleotides whichare useful as therapeutics, diagnostics, and research reagents.Sugar-modified oligonucleotides which are resistant to nucleasedegradation and are capable of modulating the activity of DNA and RNAare provided.

BACKGROUND OF THE INVENTION

It has been recognized that oligonucleotides can be used to modulatemRNA expression by a mechanism that involves the complementaryhybridization of relatively short oligonucleotides to mRNA such that thenormal, essential functions of these intracellular nucleic acids aredisrupted. Hybridization is the sequence-specific base pair hydrogenbonding of an oligonucleotide to a complementary RNA or DNA.

One deficiency of oligonucleotides for these purposes is theirsusceptibility to enzymatic degradation by a variety of ubiquitousnucleases which may be intracellularly and extracellularly located.Unmodified, “wild type”, oligonucleotides are not useful as therapeuticagents because they are rapidly degraded by nucleases. Therefore,modification of oligonucleotides for conferring nuclease resistance onthem has been a focus of research directed towards the development ofoligonucleotide therapeutics and diagnostics.

In addition to nuclease stability, the ability of an oligonucleotide tobind to a specific DNA or RNA with fidelity is a further importantfactor.

The relative ability of an oligonucleotide to bind to complementarynucleic acids is compared by determining the melting temperature of aparticular hybridization complex. The melting temperature (T_(m)), acharacteristic physical property of double helices, is the temperature(in ° C.) at which 50% helical versus coil (unhybridized) forms arepresent. T_(m) is measured by using UV spectroscopy to determine theformation and breakdown (melting) of hybridization. Base stacking, whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the binding of the nucleic acid strands.

Therefore, oligonucleotides modified to exhibit resistance to nucleasesand to hybridize with appropriate strength and fidelity to its targetedRNA (or DNA) are greatly desired for use as research reagents,diagnostic agents and as oligonucleotide therapeutics. Various2′-substitutions have been introduced in the sugar moiety ofoligonucleotides. The nuclease resistance of these compounds has beenincreased by the introduction of 2′-substituents such as halo, alkoxyand allyloxy groups.

Ikehara et al. [European Journal of Biochemistry 139, 447 (1984)] havereported the synthesis of a mixed octamer containing one2′-deoxy-2′-fluoroguanosine residue or one 2′-deoxy-2′-fluoroadenineresidue. Guschlbauer and Jankowski [Nucleic Acids Res. 8, 1421 (1980)]have shown that the contribution of the 3′-endo increases withincreasing electronegativity of the 2′-substituent. Thus,2′-deoxy-2′-fluorouridine contains 85% of the C3′-endo conformer.

Furthermore, evidence has been presented which indicates that2′-substituted-2′-deoxyadenosine polynucleotides resembledouble-stranded RNA rather than DNA. Ikehara et al. [Nucleic Acids Res.,5, 3315 (1978)] have shown that a 2′-fluoro substituent in poly A, polyI, or poly C duplexed to its complement is significantly more stablethan the ribonucleotide or deoxyribonucleotide poly duplex as determinedby standard melting assays. Ikehara et al. [Nucleic Acids Res., 4, 4249(1978)] have shown that a 2′-chloro or bromo substituent inpoly(2′-deoxyadenylic acid) provides nuclease resistance. Eckstein etal. [Biochemistry, 11, 4336 (1972)] have reported thatpoly(2′-chloro-2′-deoxyuridylic acid) andpoly(2′-chloro-2′-deoxycytidylic acid) are resistant to variousnucleases. Inoue et al. [Nucleic Acids Research, 15, 6131 (1987)] havedescribed the synthesis of mixed oligonucleotide sequences containing2′-OMe substituents on every nucleotide. The mixed 2′-OMe-substitutedoligonucleotide hybridized to its RNA complement as strongly as theRNA-RNA duplex which is significantly stronger than the same sequenceRNA-DNA heteroduplex (T_(m)s, 49.0 and 50.1 versus 33.0 degrees fornonamers). Shibahara et al. [Nucleic Acids Research, 17, 239 (1987)]have reported the synthesis of mixed oligonucleotides containing 2′-OMesubstituents on every nucleotide. The mixed 2′-OMe-substitutedoligonucleotides were designed to inhibit HIV replication.

It is believed that the composite of the hydroxyl group's steric effect,its hydrogen bonding capabilities, and its electronegativity versus theproperties of the hydrogen atom is responsible for the gross structuraldifference between RNA and DNA. Thermal melting studies indicate thatthe order of duplex stability (hybridization) of 2′-methoxyoligonucleotides is in the order of RNA-RNA>RNA-DNA>DNA-DNA.

U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixedoligonucleotides comprising an RNA portion, bearing 2′-O-alkylsubstituents, conjugated to a DNA portion via a phosphodiester linkage.However, being phosphodiesters, these oligonucleotides are susceptibleto nuclease cleavage.

European Patent application 339,842, filed Apr. 13, 1989, discloses2′-O-substituted phosphorothioate oligonucleotides, including2′-O-methylribooligonucleotide phosphorothioate derivatives. Thisapplication also discloses 2′-O-methyl phosphodiester oligonucleotideswhich lack nuclease resistance.

European Patent application 260,032, filed Aug. 27, 1987, disclosesoligonucleotides having 2′-O-methyl substituents on the sugar moiety.This application also makes mention of other 2′-O-alkyl substituents,such as ethyl, propyl and butyl groups.

International Publication Number WO 91/06556, published May 16, 1991,discloses oligomers derivatized at the 2′ position with substituents,which are stable to nuclease activity. Specific 2′-O-substituents whichwere incorporated into oligonucleotides include ethoxycarbonylmethyl(ester form), and its acid, amide and substituted amide forms.

European Patent application 399,330, filed May 15, 1990, disclosesnucleotides having 2′-O-alkyl substituents.

International Publication Number WO 91/15499, published Oct. 17, 1991,discloses oligonucleotides bearing 2′-O-alkyl, -alkenyl and -alkynylsubstituents.

It has been recognized that nuclease resistance of oligonucleotides andfidelity of hybridization are of great importance in the development ofoligonucleotide therapeutics. Oligonucleotides possessing nucleaseresistance are also desired as research reagents and diagnostic agents.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, compositions which areresistant to nuclease degradation and those that modulate the activityof DNA and RNA are provided. These compositions are comprised ofsugar-modified oligonucleotides, which are specifically hybridizablewith preselected nucleotide sequences of single-stranded ordouble-stranded target DNA or RNA. The sugar-modified oligonucleotidesrecognize and form double strands with single-stranded DNA and RNA.

The nuclease resistant oligonucleotides of the present invention consistof a single strand of nucleic acid bases linked together through linkinggroups. The oligonucleotides of this invention may range in length fromabout 5 to about 50 nucleic acid bases. However, in accordance with apreferred embodiment of this invention, a sequence of about 12 to 25bases in length is optimal.

The individual nucleotides of the oligonucleotides of the presentinvention are connected via phosphorus linkages. Preferred phosphorouslinkages include phosphodiester, phosphorothioate and phosphorodithioatelinkages, with phosphodiester and phosphorothioate linkages beingparticularly preferred.

Preferred nucleobases of the invention include adenine, guanine,cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halocytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil,4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine,8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substitutedguanines, other aza and deaza uracils, other aza and deaza thymidines,other aza and deaza cytosines, other aza and deaza adenines, other azaand deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

In accordance with this invention at least one of the2′-deoxyribofuranosyl moiety of at least one of the nucleosides of anoligonucleotide is modified. A halo, alkoxy, aminoalkoxy, alkyl, azido,or amino group may be added. For example, F, CN, CF₃, OCF₃, OCN,O-alkyl, S-alkyl, SMe, SO₂Me, ONO₂, NO₂, NH₃, NH₂, NH-alkyl, OCH₂CH═CH₂(allyloxy), OCH₃═CH₂, OCCH, where alkyl is a straight or branched chainof C₁ to C₂₀, with unsaturation within the carbon chain.

The present invention also includes oligonucleotides formed from aplurality of linked-β-nucleosides including2′-deoxy-erythro-pentofuranosyl-β-nucleosides. These nucleosides areconnected by charged phosphorus linkages in a sequence that isspecifically hybridizable with a complementary target nucleic acid. Thesequence of linked nucleosides is divided into at least twosubsequences. The first subsequence includes β-nucleosides, having2′-substituents, linked by charged 3′-5′ phosphorous linkages. Thesecond subsequence consists of2′-deoxy-erythro-pentofuranosyl-β-nucleosides linked by charged 3′-5′phosphorous linkages bearing a negative charge at physiological pH. Infurther preferred embodiments there exists a third subsequence whosenucleosides are selected from those selectable for the firstsubsequence. In preferred embodiments the second subsequence ispositioned between the first and third subsequences. Sucholigonucleotides of the present invention are also referred to as“chimeric” or “gapped” oligonucleotides, or “chimeras.”

The resulting novel oligonucleotides of the invention are resistant tonuclease degradation and exhibit hybridization properties of higherquality relative to wild-type DNA-DNA and RNA-DNA duplexes andphosphorus-modified oligonucleotide duplexes containingmethylphosphonates, phosphoramidates and phosphate triesters.

The invention is also directed to methods for modulating the productionof a protein by an organism comprising contacting the organism with acomposition formulated in accordance with the foregoing considerations.It is preferred that the RNA or DNA portion which is to be modulated bepreselected to comprise that portion of DNA or RNA which codes for theprotein whose formation is to be modulated. Therefore, theoligonucleotide to be employed is designed to be specificallyhybridizable to the preselected portion of target DNA or RNA.

This invention is also directed to methods of treating an organismhaving a disease characterized by the undesired production of a protein.This method comprises contacting the organism with a composition inaccordance with the foregoing considerations. The composition ispreferably one which is designed to specifically bind with mRNA whichcodes for the protein whose production is to be inhibited.

The invention further is directed to diagnostic methods for detectingthe presence or absence of abnormal RNA molecules, or abnormal orinappropriate expression of normal RNA molecules in organisms or cells.

The invention is also directed to methods for the selective binding ofRNA for use as research reagents and diagnostic agents. Such selectiveand strong binding is accomplished by interacting such RNA or DNA witholigonucleotides of the invention which are resistant to degradativenucleases and which display greater fidelity of hybridization than anyother known oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing dose response activity of oligonucleotides ofthe invention and a reference compound.

FIG. 2 is a bar chart showing dose response activity of oligonucleotidesof the invention and reference compounds.

FIG. 3 is a bar graph showing the effects of several 2′-O-methylchimeric oligonucleotides on PKC-α mRNA levels. Hatched bars representthe 8.5 kb transcript, and plain bars represent the 4.0 kb transcript.

FIG. 4 is a bar graph showing the effects of several 2′-O-methyl and2′-O-propyl chimeric oligonucleotides on PKC-A mRNA levels. Hatched barsrepresent the 8.5 kb transcript, and plain bars represent the 4.0 kbtranscript.

FIG. 5 is a bar graph showing the effects of additional 2′-O-methyl and2′-O-propyl chimeric oligonucleotides on PKC-α mRNA levels. Hatched barsrepresent the 8.5 kb transcript, and plain bars represent the 4.0 kbtranscript.

FIG. 6 is a graph showing mouse plasma concentrations of a controlcompound and two of the compounds of the invention. The plasmaconcentration is plotted verses time.

FIG. 7 is a three dimensional graph showing distribution of a controlcompound among various tissue in the mouse. Specific tissues are shownon one axis, time on a second axis and percent of dose on the thirdaxis. The compound was delivered by intravenous injected.

FIG. 8 is a three dimensional graph showing distribution of a compoundof the invention among various tissue in the mouse. Specific tissues areshown on one axis, time on a second axis and percent of dose on thethird axis. The compound was delivered by intravenous injected.

FIG. 9 is a three dimensional graph showing distribution of a furthercompound of the invention among various tissue in the mouse. Specifictissues are shown on one axis, time on a second axis and percent of doseon the third axis. The compound was delivered by intravenous injected.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The compositions useful for modulating the activity of an RNA or DNAmolecule in accordance with this invention generally comprise asugar-modified oligonucleotide which is specifically hybridizable with apreselected nucleotide sequence of a single-stranded or double-strandedtarget DNA or RNA molecule, and which is nuclease resistant.

It is generally desirable to select a sequence of DNA or RNA which isinvolved in the production of a protein whose synthesis is ultimately tobe modulated or inhibited in its entirety. The oligonucleotides of theinvention are conveniently synthesized using solid phase synthesis ofknown methodology, and is designed to be complementary to orspecifically hybridizable with the preselected nucleotide sequence ofthe target RNA or DNA. Nucleic acid synthesizers are commerciallyavailable and their use is understood by persons of ordinary skill inthe art as being effective in generating any desired oligonucleotide ofreasonable length.

The oligonucleotides of the invention also include those that comprisenucleosides connected by charged linkages, and whose sequences aredivided into at least two subsequences. The first subsequence includes2′-substituted-nucleosides linked by a first type of linkage. The secondsubsequence includes nucleosides linked by a second type of linkage. Ina preferred embodiment there exists a third subsequence whosenucleosides are selected from those selectable for the firstsubsequence, and the second subsequence is positioned between the firstand the third subsequences. Such oligonucleotides of the invention areknown as “chimeras,” or “chimeric” or “gapped” oligonucleotides.

In the context of this invention, the term “oligonucleotide” refers to aplurality of nucleotides joined together in a specific sequence fromnaturally and non-naturally occurring nucleobases. Preferred nucleobasesof the invention are joined through a sugar moiety via phosphoruslinkages, and include adenine, guanine, adenine, cytosine, uracil,thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl andother alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil,6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines,8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxylguanine and other 8-substituted guanines, other aza and deaza uracils,other aza and deaza thymidines, other aza and deaza cytosines, other azaand deaza adenines, other aza and deaza guanines, 5-trifluoromethyluracil and 5-trifluoro cytosine. The sugar moiety may be deoxyribose orribose. The oligonucleotides of the invention may also comprise modifiednucleobases or nucleobases having other modifications consistent withthe spirit of this invention, and in particular modifications thatincrease their nuclease resistance in order to facilitate their use astherapeutic, diagnostic or research reagents.

The oligonucleotides of the present invention are about 5 to about 50bases in length. It is more preferred that the oligonucleotides of theinvention have from 8 to about 40 bases, and even more preferred thatfrom about 12 to about 25 bases be employed.

It is desired that the oligonucleotides of the invention be adapted tobe specifically hybridizable with the nucleotide sequence of the targetRNA or DNA selected for modulation. Oligonucleotides particularly suitedfor the practice of one or more embodiments of the present inventioncomprise 2′-sugar modified oligonucleotides wherein one or more of the2′-deoxy ribofuranosyl moieties of the nucleoside is modified with ahalo, alkoxy, aminoalkoxy, alkyl, azido, or amino group. For example,the substitutions which may occur include F, CN, CF₃, OCF₃, OCN,O-alkyl, S-alkyl, SMe, SO₂Me, ONO₂, NO₂, NH₃, NH₂, NH-alkyl, OCH₃═CH₂and OCCH. In each of these, alkyl is a straight or branched chain of C₁to C₂₀, having unsaturation within the carbon chain. A preferred alkylgroup is C₁-C₉ alkyl. A further preferred alkyl group is C₅-C₂₀ alkyl.

A first preferred group of substituents include 2′-deoxy-2′-fluorosubstituents. A further preferred group of substituents include C₁-C₂₀alkoxyl substituents. An additional preferred group of substituentsinclude cyano, fluoromethyl, thioalkoxyl, fluoroalkoxyl, alkylsulfinyl,alkylsulfonyl, allyloxy and alkeneoxy substituents.

In further embodiments of the present invention, the individualnucleotides of the oligonucleotides of the invention are connected viaphosphorus linkages. Preferred phosphorus linkages includephosphodiester, phosphorothioate and phosphorodithioate linkages. In onepreferred embodiment of this invention, nuclease resistance is conferredon the oligonucleotides by utilizing phosphorothioate internucleosidelinkages.

In further embodiments of the invention, nucleosides can be joined vialinkages that substitute for the internucleoside phosphate linkage.Macromolecules of this type have been identified as oligonucleosides.The term “oligonucleoside” thus refers to a plurality of nucleosideunits joined by non-phosphorus linkages. In such oligonucleosides thelinkages include an —O—CH₂—CH₂—O— linkage (i.e., an ethylene glycollinkage) as well as other novel linkages disclosed in U.S. Pat. No.5,223,618, issued Jun. 29, 1993, U.S. Pat. No. 5,378,825, issued Jan. 3,1995 and U.S. patent application Ser. No. 08/395,168, filed Feb. 27,1995. Other modifications can be made to the sugar, to the base, or tothe phosphate group of the nucleotide. Representative modifications aredisclosed in International Publication Numbers WO 91/10671, publishedJul. 25, 1991, WO 92/02258, published Feb. 20, 1992, WO 92/03568,published Mar. 5, 1992, and U.S. Pat. No. 5,138,045, issued Aug. 11,1992, all assigned to the assignee of this application. The disclosuresof each of the above referenced publications are herein incorporated byreference.

In the context of this invention, “hybridization” shall mean hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleotides. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. “Complementary,” as used herein, alsorefers to sequence complementarity between two nucleotides. For example,if a nucleotide at a certain position of an oligonucleotide is capableof hydrogen bonding with a nucleotide at the same position of a DNA orRNA molecule, then the oligonucleotide and the DNA or RNA are consideredto be complementary to each other at that position. The oligonucleotideand the DNA or RNA are complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between the oligonucleotide and the DNA or RNA target. Itis understood that an oligonucleotide need not be 100% complementary toits target DNA sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA, and there is a sufficientdegree of complementarity to avoid non-specific binding of theoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e. under physiological conditions in thecase of in vivo assays or therapeutic treatment, or in the case of invitro assays, under conditions in which the assays are performed.

Cleavage of oligonucleotides by nucleolytic enzymes require theformation of an enzyme-substrate complex, or in particular anuclease-oligonucleotide complex. The nuclease enzymes will generallyrequire specific binding sites located on the oligonucleotides forappropriate attachment. If the oligonucleotide binding sites are removedor blocked, such that nucleases are unable to attach to theoligonucleotides, the oligonucleotides will be nuclease resistant. Inthe case of restriction endonucleases that cleave sequence-specificpalindromic double-stranded DNA, certain binding sites such as the ringnitrogen in the 3- and 7-positions have been identified as requiredbinding sites. Removal of one or more of these sites or stericallyblocking approach of the nuclease to these particular positions withinthe oligonucleotide has provided various levels of resistance tospecific nucleases.

This invention provides oligonucleotides possessing superiorhybridization properties. Structure-activity relationship studies haverevealed that an increase in binding (T_(m)) of certain 2′-sugarmodified oligonucleotides to an RNA target (complement) correlates withan increased “A” type conformation of the heteroduplex. Furthermore,absolute fidelity of the modified oligonucleotides is maintained.Increased binding of 2′-sugar modified sequence-specificoligonucleotides of the invention provides superior potency andspecificity compared to phosphorus-modified oligonucleotides such asmethyl phosphonates, phosphate triesters and phosphoramidates as knownin the literature.

The only structural difference between DNA and RNA duplexes is ahydrogen atom at the 2′-position of the sugar moiety of a DNA moleculeversus a hydroxyl group at the 2′-position of the sugar moiety of an RNAmolecule (assuming that the presence or absence of a methyl group in theuracil ring system has no effect). However, gross conformationaldifferences exist between DNA and RNA duplexes.

It is known from X-ray diffraction analysis of nucleic acid fibers[Arnott and Hukins, Biochemical and Biophysical Research Communication,47, 1504-1510 (1970)] and analysis of crystals of double-strandednucleic acids that DNA takes a “B” form structure and RNA takes the morerigid “A” form structure. The difference between the sugar puckering(C2′ endo for “B” form DNA and C3′ endo for “A” form RNA) of thenucleosides of DNA and RNA is the major conformational differencebetween double-stranded nucleic acids.

The primary contributor to the conformation of the pentofuranosyl moietyis the nature of the substituent at the 2′-position. Thus, thepopulation of the C3′-endo form increases with respect to the C2′-endoform as the electronegativity of the 2′-substituent increases. Forexample, among 2′-deoxy-2′-haloadenosines, the 2′-fluoro derivativeexhibits the largest population (65%) of the C3′-endo form, and the2′-iodo exhibits the lowest population (7%). Those of adenosine (2′-OH)and deoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore,the effect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoroadenosine) is furthercorrelated to the stabilization of the stacked conformation. Researchindicates that dinucleoside phosphates have a stacked conformation witha geometry similar to that of A-A but with a greater extent of base-baseoverlapping than A-A. It is assumed that the highly polar nature of theC2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an “A” structure.

Data from UV hypochromicity, circular dichroism, and ¹H NMR alsoindicate that the degree of stacking decreases as the electronegativityof the halo substituent decreases. Furthermore, steric bulk at the2′-position of the sugar moiety is better accommodated in an “A” formduplex than a “B” form duplex.

Thus, a 2′-substituent on the 3′-nucleotidyl unit of a dinucleosidemonophosphate is thought to exert a number of effects on the stackingconformation: steric repulsion, furanose puckering preference,electrostatic repulsion, hydrophobic attraction, and hydrogen bondingcapabilities. These substituent effects are thought to be determined bythe molecular size, electronegativity, and hydrophobicity of thesubstituent.

The 2′-iodo substituted nucleosides possess the lowest C3′-endopopulation (7%) of the halogen series. Thus, based solely on stericeffects, one would predict that a 2′-iodo (or other similar group) wouldcontribute stacking destabilization properties, and thus reduced binding(T_(m)) of the oligonucleotides. However, the lower electronegativityand high hydrophobicity of the iodine atom (or another similar group)complicates the ability to predict stacking stabilities and bindingstrengths.

Studies with a 2′-OMe modification of 2′-deoxy guanosine, cytidine, anduridine dinucleoside phosphates exhibit enhanced stacking effects withrespect to the corresponding unmethylated species (2′-0H). In this case,the hydrophobic attractive forces of the methyl group tend to overcomethe destabilizing effects of its steric bulk.

2′-Fluoro-2′-deoxyadenosine has been determined to have an unusuallyhigh population of 3′-endo puckered form among nucleosides. Adenosine,2′-deoxyadenosine and other derivatives have less than 40% of theirpopulation in the 3′-endo conformation. It is known that a nucleosideresidue in well-stacked oligonucleotides favors 3′-endo ribofuranosepuckering.

Melting temperatures (complementary binding) are increased with the2′-substituted adenosine diphosphates. It is not clear whether the3′-endo preference of the conformation or the presence of thesubstituent is responsible for the increased binding. However, greateroverlap of adjacent bases (stacking) can be achieved with the 3′-endoconformation.

Compounds of the invention can be utilized as diagnostics, therapeuticsand as research reagents and kits. They can be utilized inpharmaceutical compositions by adding an effective amount of anoligonucleotide of the invention to a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism can be contacted with an oligonucleotide of theinvention having a sequence that is capable of specifically hybridizingwith a strand of target nucleic acid that codes for the undesirableprotein.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered an oligomer in accordance with the invention, commonly in apharmaceutically acceptable carrier, in doses ranging from 0.01 μg to100 g per kg of body weight depending on the age of the patient and theseverity of the disease state being treated. Further, the treatment maybe a single dose or may be a regimen that may last for a period of timewhich will vary depending upon the nature of the particular disease, itsseverity and the overall condition of the patient, and may extend fromonce daily to once every 20 years. Following treatment, the patient ismonitored for changes in his/her condition and for alleviation of thesymptoms of the disease state. The dosage of the oligomer may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disease state is observed, or if the disease statehas been ablated.

In some cases it may be more effective to treat a patient with anoligomer of the invention in conjunction with other traditionaltherapeutic modalities. For example, a patient being treated for AIDSmay be administered an oligomer in conjunction with AZT, or a patientwith atherosclerosis may be treated with an oligomer of the inventionfollowing angioplasty to prevent reocclusion of the treated arteries.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual oligomers, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to several years.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the oligomer is administered in maintenance doses,ranging from 0.01 μg to 100 g per kg of body weight, once or more daily,to once every several years.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

The present invention can be practiced in a variety of organisms rangingfrom unicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular machinery is susceptible to such therapeuticand/or prophylactic treatment. Seemingly diverse organisms such asbacteria, yeast, protozoa, algae, plant and higher animal forms,including warm-blooded animals, can be treated in this manner. Further,since each of the cells of multicellular eukaryotes also includes bothDNA-RNA transcription and RNA-protein translation as an integral part oftheir cellular activity, such therapeutics and/or diagnostics can alsobe practiced on such cellular populations. Furthermore, many of theorganelles, e.g. mitochondria and chloroplasts, of eukaryotic cells alsoinclude transcription and translation mechanisms. As such, single cells,cellular populations or organelles also can be included within thedefinition of organisms that are capable of being treated with thetherapeutic or diagnostic oligonucleotides of the invention. As usedherein, therapeutics is meant to include both the eradication of adisease state, killing of an organism, e.g. bacterial, protozoan orother infection, or control of aberrant or undesirable cellular growthor expression.

The present novel approach to obtaining stronger binding is to prepareRNA mimics that bind to target RNA. Therefore, a randomstructure-activity relationship approach was undertaken to discovernuclease resistant oligonucleotides that maintain appropriatehybridization properties.

A series of 2′-deoxy-2′-modified nucleosides of adenine, guanine,cytosine, thymidine and certain analogs of these nucleobases have beenprepared and incorporated into oligonucleotides via solid phase nucleicacid synthesis. These novel oligonucleotides were assayed for theirhybridization properties and their ability to resist degradation bynucleases compared to the unmodified oligonucleotides. Initially, smallelectronegative atoms or groups were selected because they would not beexpected to sterically interfere with required Watson-Crick base pairhydrogen bonding (hybridization). However, electronic changes due to theelectronegativity of the atom or group in the 2′-position may profoundlyaffect the sugar conformation. Structure-activity relationship studiesrevealed that the sugar-modified oligonucleotides hybridized to thetarget RNA more strongly than the unmodified 2′-deoxy oligonucleotides.

2′-Substituted oligonucleotides were synthesized by standard solid phasenucleic acid synthesis using an automated synthesizer such as Model 380B(Perkin-Elmer/Applied Biosystems) or MilliGen/Biosearch 7500 or 8800.Triester, phosphoramidite, or hydrogen phosphonate coupling chemistries[Oligonucleotides. Antisense Inhibitors of Gene Expression. M.Caruthers, p. 7, J. S. Cohen (Ed.), CRC Press, Boca Raton, Fla., 1989]are used with these synthesizers to provide the desiredoligonucleotides. The Beaucage reagent [J. Amer. Chem. Soc., 112, 1253(1990)] or elemental sulfur [Beaucage et al., Tet. Lett., 22, 1859(1981)] is used with phosphoramidite or hydrogen phosphonate chemistriesto provide 2′-substituted phosphorothioate oligonucleotides.

The requisite 2′-substituted nucleosides (A, G, C, T(U), and othermodified nucleobases) were prepared by modification of severalliterature procedures as described below.

Procedure 1. Nucleophilic Displacement of 2′-Leaving Group in ArabinoPurine Nucleosides. Nucleophilic displacement of a leaving group in the2′-up position (2′-deoxy-2′-(leaving group)arabino sugar) of adenine orguanine or their analog nucleosides. General synthetic procedures ofthis type have been described by Ikehara et al., Tetrahedron, 34, 1133(1978); ibid., 31, 1369 (1975); Chemistry and Pharmaceutical Bulletin,26, 2449 (1978); ibid., 26, 240 (1978); Ikehara, Accounts of ChemicalResearch, 2, 47 (1969); and Ranganathan, Tetrahedron Letters, 15, 1291(1977).

Procedure 2. Nucleophilic Displacement of 2,2′-Anhydro Pyrimidines.Nucleosides thymine, uracil, cytosine or their analogs are converted to2′-substituted nucleosides by the intermediacy of 2,2′-cycloanhydronucleoside as described by Fox et al., Journal of Organic Chemistry, 29,558 (1964).

Procedure 3. 2′-Coupling Reactions. Appropriately 3′,5′-sugar and baseprotected purine and pyrimidine nucleosides having a unprotected2′-hydroxyl group are coupled with electrophilic reagents such as methyliodide and diazomethane to provide the mixed sequences containing a2′-OMe group H. Inoue et al., Nucleic Acids Research, 15, 6131.

Procedure 4. 2-Deoxy-2-substituted Ribosylations.2-Substituted-2-deoxyribosylation of the appropriately protected nucleicacid bases and nucleic acids base analogs has been reported by Jarvi etal., Nucleosides & Nucleotides, 8, 1111-1114 (1989) and Hertel et al.,Journal of Organic Chemistry, 53, 2406 (1988).

Procedure 5. Enzymatic Synthesis of 2′-Deoxy-2′-Substituted Nucleosides.The 2-Deoxy-2-substituted glycosyl transfer from one nucleoside toanother with the aid of pyrimidine and purine ribo or deoxyribophosphorolyses has been described by Rideout and Krenitsky, U.S. Pat.No. 4,381,344 (1983).

Procedure 6. Conversion of 2′-Substituents Into New Substituents.2′-Substituted-2′-deoxynucleosides are converted into new substituentsvia standard chemical manipulations. For example, Chladek et al.[Journal of Carbohydrates, Nucleosides & Nucleotides, 7, 63 (1980)]describes the conversion of 2′-deoxy-2′-azidoadenosine, prepared fromarabinofuranosyladenine, into 2′-deoxy-2′-aminoadenosine.

Procedure 7. Free Radical Reactions. Conversions of halogen substitutednucleosides into 2′-deoxy-2′-substituted nucleosides via free radicalreactions has been described by Parkes and Taylor [Tetrahedron Letters,29, 2995 (1988)].

Procedure 8. Conversion of Ribonucleosides to 2′-Deoxy-2′-SubstitutedNucleoside. Appropriately 3′,5′-sugar and base protected purine andpyrimidine nucleosides having a unprotected 2′-hydroxyl group areconverted to 2′-deoxy-2′-substituted nucleosides by the process ofoxidation to the 2′-keto group, reaction with nucleophilic reagents, andfinally 2′-deoxygenation. Procedures of this type have been described byDe las Heras, et al. [Tetrahedron Letters, 29, 941 (1988)].

Procedure 9. In one process of the invention, 2′-deoxy substitutedguanosine compounds are prepared via an (arabinofuranosyl)guanineintermediate obtained via an oxidation-reduction reaction. A leavinggroup at the 2′ position of the arabinofuranosyl sugar moiety of theintermediate arabino compound is displaced via an SN₂ reaction with anappropriate nucleophile. This procedure thus incorporates principles ofboth Procedure 1 and Procedure 8 above. 2′-Deoxy-2′-fluoroguanosine ispreferably prepared via this procedure. The intermediate arabinocompound was obtained utilizing a variation of the oxidation-reductionprocedure of Hansske et al. [Tetrahedron, 40, 125 (1984)]. According tothis invention, the reduction was effected starting at −78° C. andallowing the reduction reaction to exothermically warm to about −2° C.This results in a high yield of the intermediate arabino compound.

In conjunction with use of a low temperature reduction, utilization of atetraisopropyldisiloxane blocking group (a “TPDS” group) for the 3′ and5′ positions of the starting guanosine compound contributes to animproved ratio of intermediate arabino compound to the ribo compoundfollowing oxidation and reduction. Following oxidation and reduction,the N² guanine amino nitrogen and the 2′-hydroxyl moieties of theintermediate arabino compound are blocked with isobutyryl protectinggroups (“Ibu” groups). The tetraisopropyldisiloxane blocking group isremoved and the 3′ and 5′ hydroxy groups are further protected with asecond blocking group, a tetrahydropyranyl blocking group (“THP” group).The isobutyryl group is selectively removed from 2′-hydroxyl groupfollowed by derivation of the 2′ position with a triflate leaving group.The triflate group was then displaced with inversion about the 2′position to yield the desired 2′-deoxy-2′-fluoroguanosine compound.

In addition to the triflate leaving group, other leaving groups include,but are not limited to, alkylsulfonyl, substituted alkylsulfonyl,arylsulfonyl, substituted arylsulfonyl, heterocyclosulfonyl ortrichloroacetimidate. Representative examples includep-(2,4-dinitroanilino)-benzenesulfonyl, benzenesulfonyl, methylsulfonyl,p-methyl-benzenesulfonyl, p-bromobenzenesulfonyl, trichloroacetimidate,acyloxy, 2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl and2,4,6-trichlorophenyl.

The isobutyryl group remaining on the N² heterocyclic amino moiety ofthe guanine ring can be removed to yield a completely deblockednucleoside. However, preferably, for incorporation of the2′-deoxy-2′-substituted compound into an oligonucleotide, deblocking ofthe 2 isobutyryl protecting group is deferred until afteroligonucleotide synthesis is complete. Normally for use in automatednucleic acid synthesizers, blocking of the N² guanine moiety with anisobutyryl group is preferred. Thus, advantageously, theN²-isobutyryl-blocked 2′-deoxy-2′-substituted guanosine compoundsresulting from the method of the invention can be directly used foroligonucleotide synthesis on automated nucleic acid synthesizers.

For the purpose of illustration, the oligonucleotides of the inventionhave been used in a ras-luciferase fusion system using ras-luciferasetransactivation. As described in International Publication Number WO92/22651, published Dec. 23, 1992 and commonly assigned with thisapplication, the entire contents of which are herein incorporated byreference, the ras oncogenes are members of a gene family that encoderelated proteins that are localized to the inner face of the plasmamembrane. Ras proteins have been shown to be highly conserved at theamino acid level, to bind GTP with high affinity and specificity, and topossess GTPase activity. Although the cellular function of ras geneproducts is unknown, their biochemical properties, along with theirsignificant sequence homology with a class of signal-transducingproteins known as GTP binding proteins, or G proteins, suggest that rasgene products play a fundamental role in basic cellular regulatoryfunctions relating to the transduction of extracellular signals acrossplasma membranes.

Three ras genes, designated H-ras, K-ras, and N-ras, have beenidentified in the mammalian genome. Mammalian ras genes acquiretransformation-inducing properties by single point mutations withintheir coding sequences. Mutations in naturally occurring ras oncogeneshave been localized to codons 12, 13, and 61. The most commonly detectedactivating ras mutation found in human tumors is in codon-12 of theH-ras gene in which a base change from GGC to GTC results in aglycine-to-valine substitution in the GTPase regulatory domain of theras protein product. This single amino acid change is thought to abolishnormal control of ras protein function, thereby converting a normallyregulated cell protein to one that is continuously active. It isbelieved that such deregulation of normal ras protein function isresponsible for the transformation from normal to malignant growth.

The oligonucleotides of the present invention have also been used formodulating the expression of the raf gene, a naturally present cellulargene which occasionally converts to an activated form that has beenimplicated in abnormal cell proliferation and tumor formation.

The oligonucleotides of the present invention are also specificallyhybridizable with nucleic acids relating to protein kinase C (PKC).These oligonucleotides have been found to modulate the expression ofPKC.

The following examples illustrate the present invention and are notintended to limit the same.

Example 1 Preparation of 2′-Deoxy-2′-fluoro Modified Oligonucleotides A.N⁶-Benzoyl-[2′-deoxy-2′-fluoro-5′-O-(4,4′-di-methoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite

N⁶-Benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine was prepared from9-β-D-arabinofuranosyladenine in a five-step synthesis using amodification of a procedure reported by Ikehara et al. [Nucleosides andNucleotides, 2, 373-385 (1983)]. The N⁶-benzoyl derivative was obtainedin good yield utilizing the method of transient protection withchlorotrimethylsilane. Jones [J. Am. Chem. Soc., 104, 1316 (1982)].Selective protection of the 3′ and 5′-hydroxyl groups ofN⁶-Benzoyl-9-β-D-arabinofuranosyladenine with tetrahydropyranyl (THP)was accomplished by modification of the literature procedure accordingto Butke et al. [Nucleic Acid Chemistry, Part 3, p. 149, L. B. Townsendand R. S. Tipson, Eds., J. Wiley and Sons, New York, 1986], to yieldN⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabino-furanosyl]adeninein good yield. Treatment ofN⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabino-furanosyl]adeninewith trifluoromethanesulfonic anhydride in dichloromethane gave the2′-triflate derivativeN⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adeninewhich was not isolated due to its lability. Displacement of the2′-triflate group was effected by reaction with tetrabutylammoniumfluoride in tetrahydrofuran to obtain a moderate yield of the 2′-fluoroderivativeN⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine.Deprotection of the THP groups ofN⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydro-pyran-2-yl)-β-D-arabinofuranosyl]adeninewas accomplished by treatment with Dowex-50W in methanol to yieldN⁶-benzoyl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)adenine in moderateyield. The ¹H-NMR spectrum was in agreement with the literature values.[Ikehara and Miki, Chem. Pharm. Bull., 26, 2449 (1978)]. Standardmethodologies were employed to obtain the5′-dimethoxytrityl-3′-phosphoramidite intermediatesN⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribo-furanosyl]adenineandN⁶-Benzoyl-[2′-deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite.[Ogilvie, Can J. Chem., 67, 831-839 (1989)].

B. N⁶-Benzoyl-9-β-D-arabinofuranosyladenine

9-β-D-arabinofuranosyladenine (1.07 g, 4.00 mmol) was dissolved inanhydrous pyridine (20 mL) and anhydrous dimethylformamide (20 mL) underan argon atmosphere. The solution was cooled to 0° C. andchlorotrimethylsilane (3.88 mL, 30.6 mmol) was added slowly to thereaction mixture via a syringe. After stirring the reaction mixture at0° C. for 30 minutes, benzoyl chloride (2.32 mL, 20 mmol) was addedslowly. The reaction mixture was allowed to warm to 20° C. and stirredfor 2 hours. After cooling the reaction mixture to 0° C., cold water (8mL) was added and the mixture was stirred for 15 minutes. Concentratedammonium hydroxide (8 mL) was slowly added to the reaction mixture togive a final concentration of 2 M of ammonia. After stirring the coldreaction mixture for 30 minutes, the solvent was evaporated in vacuo (60torr) at 20° C. followed by evaporation in vacuo (1 torr) at 40° C. togive an oil. This oil was triturated with diethyl ether (50 mL) to givea solid which was filtered and washed with diethyl ether three times.This crude solid was triturated in methanol (100 mL) at refluxtemperature three times and the solvent was evaporated to yieldN⁶-Benzoyl-9-β-D-arabino-furanosyladenine as a solid (1.50 g, 100%).

C. N⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine

N⁶-Benzoyl-9-β-D-arabinofuranosyladenine (2.62 g, 7.06 mmol) wasdissolved in anhydrous dimethylformamide (150 mL) under argon andp-toluenesulfonic acid monohydrate (1.32 g, 6.92 mmol) was added. Thissolution was cooled to 0° C. and dihydropyran (1.26 mL, 13.8 mmol) wasadded via a syringe. The reaction mixture was allowed to warm to 20° C.Over a period of 5 hours a total of 10 equivalents of dihydropyran wereadded in 2 equivalent amounts in the fashion described. The reactionmixture was cooled to 0° C. and saturated aqueous sodium bicarbonate wasadded slowly to a pH of 8, then water was added to a volume of 750 mL.The aqueous mixture was extracted with methylene chloride (4×200 mL),and the organic phases were combined and dried over magnesium sulfate.The solids were filtered and the solvent was evaporated in vacuo (60torr) at 30° C. to give a small volume of liquid which was evaporated invacuo (1 torr) at 40° C. to give an oil. This oil was coevaporated withp-xylene in vacuo at 40° C. to give an oil which was dissolved inmethylene chloride (100 mL). Hexane (200 mL) was added to the solutionand the lower-boiling solvent was evaporated in vacuo at 30° C. to leavea white solid suspended in hexane. This solid was filtered and washedwith hexane (3×10 mL) then purified by column chromatography usingsilica gel and methylene chloride-methanol (93:7) as the eluent. Thefirst fraction yielded the title compound 3 as a white foam (3.19 g,83%) and a second fraction gave a white foam (0.81 g) which wascharacterized as the 5′-monotetrahydropyranyl derivative ofN⁶-Benzoyl-9-β-D-arabinofuranosyladenine.

D.N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine

N⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(2.65 g, 4.91 mmol) was dissolved in anhydrous pyridine (20 mL) and thesolvent was evaporated in vacuo (1 mm Hg) at 40° C. The resulting oilwas dissolved in anhydrous methylene chloride (130 mL) under argonanhydrous pyridine (3.34 mL, 41.3 mmol) and N,N-dimethylaminopyridine(1.95 g, 16.0 mmol) were added. The reaction mixture was cooled to 0° C.and trifluoromethanesulfonic anhydride (1.36 mL, 8.05 mmol) was addedslowly via a syringe. After stirring the reaction mixture at 0° C. for 1hour, it was poured into cold saturated aqueous sodium bicarbonate (140mL). The mixture was shaken and the organic phase was separated and keptat 0° C. The aqueous phase was extracted with methylene chloride (2×140mL). The organic extracts which were diligently kept cold were combinedand dried over magnesium sulfate. The solvent was evaporated in vacuo(60 torr) at 20° C. then evaporated in vacuo (1 torr) at 20° C. to giveN⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenineas a crude oil which was not purified further.

E.N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydro-pyran-2-yl)-β-D-arabinofuranosyl]adenine

N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(4.9 mmol) as a crude oil was dissolved in anhydrous tetrahydrofuran(120 mL) and this solution was cooled to 0° C. under argon.Tetrabutylammonium fluoride as the hydrate (12.8 g, 49.1 mmol) wasdissolved in anhydrous tetrahydrofuran (50 mL) and half of this volumewas slowly added via a syringe to the cold reaction mixture. Afterstirring at 0° C. for 1 hour, the remainder of the reagent was addedslowly. The reaction mixture was stirred at 0° C. for an additional 1hour, then the solvent was evaporated in vacuo (60 torr) at 20° C. togive an oil. This oil was dissolved in methylene chloride (250 mL) andwashed with brine three times. The organic phase was separated and driedover magnesium sulfate. The solids were filtered and the solvent wasevaporated to give an oil. The crude product was purified by columnchromatography using silica gel in a sintered-glass funnel and ethylacetate was used as the eluent.N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adeninewas obtained as an oil (2.03 g, 76%).

F. N⁶-Benzoyl-9-(2′-fluoro-β-D-ribofuranosyl)adenine

N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(1.31 g, 2.42 mmol) was dissolved in methanol (60 mL), and Dowex50W×2-100 (4 cm³, 2.4 m. eq) was added to the reaction mixture. Thereaction mixture was stirred at 20° C. for 1 hour then cooled to 0° C.Triethylamine (5 mL) was then slowly added to the cold reaction mixtureto a pH of 12. The resin was filtered and washed with 30% triethylaminein methanol until the wash no longer contained UV absorbing material.Toluene (50 mL) was added to the washes and the solvent was evaporatedat 24° C. in vacuo (60 torr, then 1 torr) to give a residue. Thisresidue was partially dissolved in methylene chloride (30 mL) and thesolvent was transferred to a separatory funnel. The remainder of theresidue was dissolved in hot (60° C.) water and after cooling thesolvent it was also added to the separatory funnel. The biphasic systemwas extracted, and the organic phase was separated and extracted withwater (3×100 mL). The combined aqueous extracts were evaporated in vacuo(60 torr, then 1 torr Hg) at 40° C. to give an oil which was evaporatedwith anhydrous pyridine (50 mL). This oil was further dried in vacuo (1torr Hg) at 20° C. in the presence of phosphorous pentoxide overnight togive N⁶-benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine as a yellow foam(1.08 g, 100%) which contained minor impurities.

G.N⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxy-trityl)-β-D-ribofuranosyl]adenine

N⁶-Benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine (1.08 g, 2.89 mmol)which contained minor impurities was dissolved in anhydrous pyridine (20mL) under argon and dry triethylamine (0.52 mL, 3.76 mmol) was addedfollowed by addition of 4,4′-dimethoxytrityl chloride (1.13 g, 3.32mmol). After 4 hours of stirring at 20° C. the reaction mixture wastransferred to a separatory funnel and diethyl ether (40 mL) was addedto give a white suspension. This mixture was washed with water threetimes (3×10 ml), the organic phase was separated and dried overmagnesium sulfate. Triethylamine (1 ml) was added to the solution andthe solvent was evaporated in vacuo (60 torr Hg) at 20° C. to give anoil which was evaporated with toluene (20 mL) containing triethylamine(1 mL). This crude product was purified by column chromatography usingsilica gel and ethyl acetate-triethylamine (99:1) followed by ethylacetate-methanol-triethylamine (80:19:1) to give the product in twofractions. The fractions were evaporated in vacuo (60 torr, then 1 torrHg) at 20° C. to give a foam which was further dried in vacuo (1 torrHg) at 20° C. in the presence of sodium hydroxide to giveN⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]adenineas a foam (1.02 g, 52%).

H. N⁶-Benzoyl-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—N,N-diisopropyl-β-cyanoethyl phosphoramidite

N⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]adenine(1.26 g, 1.89 mmol) was dissolved in anhydrous dichloromethane (13 mL)under argon, diisopropylethylamine (0.82 mL, 4.66 mmol) was added, andthe reaction mixture was cooled to 0° C.

Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.88 mL, 4.03 mmol) wasadded to the reaction mixture which was allowed to warm to 20° C. andstirred for 3 hours. Ethylacetate (80 mL) and triethylamine (1 mL) wereadded and this solution was washed with brine (3×25 mL). The organicphase was separated and dried over magnesium sulfate. After filtrationof the solids the solvent was evaporated in vacuo at 20° C. to give anoil which was purified by column chromatography using silica gel andhexanes-ethyl acetate-triethyl-amine (50:49:1) as the eluent.Evaporation of the fractions in vacuo at 20° C. gave a foam which wasevaporated with anhydrous pyridine (20 mL) in vacuo (1 torr) at 26° C.and further dried in vacuo (1 torr Hg) at 20° C. in the presence ofsodium hydroxide for 24 h to giveN⁶-Benzoyl-[2′-deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramiditeas a foam (1.05 g, 63%).

I.2′-Deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-uridine-3′-O—(N,N-diisopropyl-β-cyanoethyl-phosphoramidite)

2,2′-Cyclouridine is treated with a solution of 70% hydrogenfluoride/pyridine in dioxane at 120° C. for ten hours to provide aftersolvent removal a 75% yield of 2′-deoxy-2′-fluorouridine. The 5′-DMT and3′-cyanoethoxydiisopropyl-phosphoramidite derivitized nucleoside isobtained by standard literature procedures [Gait, Ed., OligonucleotideSynthesis. A Practical Approach, IRL Press, Washington, D.C. (1984)], oraccording to the procedure of Example 1A.

J.2′-Deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)

2′-Deoxy-2′-fluorouridine (2.51 g, 10.3 mmol) was converted tocorresponding cytidine analog via the method of C. B. Reese, et al., J.Chem. Soc. Perkin Trans I, pp. 1171-1176 (1982), by acetylation withacetic anhydride (3.1 mL, 32.7 mmol) in anhydrous pyridine (26 mL) atroom temperature. The reaction was quenched with methanol, the solventwas evaporated in vacuo (1 torr) to give an oil which was coevaporatedwith ethanol and toluene. 3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wascrystallized from ethanol to afford colorless crystals (2.38 g, 81%).

N-4-(1,2,4-triazol-1-yl)-3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wasobtained in a 70% yield (2.37 g) by reaction of3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine (2.75 g, 9.61 mmol) with1,2,4-triazole (5.97 g, 86.5 mmol), phosphorus oxychloride (1.73 mL,18.4 mmol), and triethylamine (11.5 mL, 82.7 mmol) in anhydrousacetonitrile at room temperature. After 90 min the reaction mixture wascooled to ice temperature and triethylamine (7.98 ml, 56.9 mmol) wasadded followed by addition of water (4.0 ml). The solvent was evaporatedin vacuo (1 torr) to give an oil which was dissolved in methylenechloride and washed with saturated aqueous sodium bicarbonate. Theaqueous phase was extracted with methylene chloride twice (2×100 mL) andthe organic extracts dried with magnesium sulfate. Evaporation of thesolvent afforded an oil from which the productN-4-(1,2,4-triazol-1-yl)-3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wasobtained by crystallization from ethanol.

2′-deoxy-2′-fluorocytidine was afforded by treatment of protectedtriazol-1-yl derivative with concentrated ammonium hydroxide (4.26 mL,81.2 mmol) in dioxane at room temperature for 6 hours. After evaporationof the solvent the oil was stirred in half-saturated (at icetemperature) ammonia in methanol for 16 hours. The solvent wasevaporated and 2′-deoxy-2′-fluoro-cytidine crystallized fromethyl-acetate-methanol (v/v, 75:25) to give colorless crystals (1.24 g,75%).

N-4-benzoyl-2′-deoxy-2′-fluorocytidine was prepared by selectivebenzoylation with benzoic anhydride in anhydrous dimethylformamide, V.Bhat, et al. Nucleosides Nucleotides, Vol. 8, pp. 179-183 (1989). The5′-O-(4,4′-dimethoxytrityl)-3′-O—(N,N-diisopropyl-β-cyanoethyl-phosphoramidite)was prepared in accordance with Example 1A.

K.9-(3′,5′-[1,1,3,3-Tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine

The 3′ and 5′ positions of guanosine were protected by the addition of aTPDS (1,1,3,3-tetraisopropyldisilox-1,3-diyl) protecting group as perthe procedure of Robins et al. [Can. J. Chem., 61, 1911 (1983)]. To astirred solution of DMSO (160 mL) and acetic anhydride (20 mL) was addedthe TPDS guanosine (21 g, 0.040 mol). The reaction was stirred at roomtemperature for 36 hours and then cooled to 0° C. Cold ethanol (400 mL,95%) was added and the reaction mixture further cooled to −78° C. in adry ice/acetone bath. NaBH₄ (2.0 g, 1.32 mol. eq.) was added. Thereaction mixture was allowed to warm up to −2° C., stirred for 30minutes and again cooled to −78° C. This was repeated twice. After theaddition of NaBH₄ was complete, the reaction was stirred at 0° c. for 30minutes and then at room temperature for 1 hour. The reaction was takenup in ethyl acetate (1 L) and washed twice with a saturated solution ofNaCl. The organic layer was dried over MgSO₄ and evaporated underreduced pressure. The residue was coevaporated twice with toluene andpurified by silica gel chromatography using CH₂Cl₂-MeOH (9:1) as theeluent. Pure product (6.02 g) precipitated from the appropriate columnfractions during evaporation of these fractions, and an additional 11.49g of product was obtained as a residue upon evaporation of thefractions.

L.N²-Isobutyryl-9-(2′-O-isobutyryl-3′,5′-[1,1,3,3-tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine

9-(3′,5′-[1,1,3,3-Tetraisopropyldisilox-1,3-diyl]β-D-arabinofuranosyl)guanine(6.5 g, 0.01248 mol) was dissolved in anhydrous pyridine (156 mL) underargon. DMAP (9.15 g) was added. Isobutyric anhydride (6.12 mL) wasslowly added and the reaction mixture stirred at room temperatureovernight. The reaction mixture was poured into cold saturated NaHCO₃(156 mL) and stirred for 10 minutes. The aqueous solution was extractedthree times with ethyl acetate (156 mL). The organic phase was washedthree times with saturated NaHCO₃ and evaporated to dryness. The residuewas coevaporated with toluene and purified by silica gel columnchromatography using CH₂Cl₂-acetone (85:15) to yield 5.67 g of product.

M. N²-Isobutyryl-9-(2′-O-isobutyryl-β-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(2′-isobutyryl-3′,5′-[1,1,3,3-tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine(9.83 g, 0.01476 mol) was dissolved in anhydrous THF (87.4 mL) at roomtemperature under argon. 1 M (nBu)₄N⁺F⁻ in THF (29.52 mL, 2 eq.) wasadded and the reaction mixture stirred for 30 minutes. The reactionmixture was evaporated at room temperature and the residue purified bysilica gel column chromatography using EtOAc-MeOH (85:15) to yield 4.98g (80%) of product.

N.N²-Isobutyryl-9-(2′-O-isobutyryl-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(2′-isobutyryl-β-D-arabinofuranosyl)guanine (4.9 g) wasdissolved in anhydrous 1,4-dioxane (98 mL) at room temperature underargon. p-Toluenesulphonic acid monohydrate (0.97 g) was added followedby 3,4-dihydro-2H-pyran (DHP, 9.34 mL, 8.8 eq.). The reaction mixturewas stirred for 2 hours, then cooled to 0° C. and saturated NaHCO₃ (125mL) was added to quench the reaction. The reaction mixture was extractedthree times with 125 mL portions of CH₂Cl₂ and the organic phase driedover MgSO₄. The organic phase was evaporated and the residue dissolvedin minimum volume of CH₂Cl₂, but in an amount sufficient to yield aclear liquid not a syrup, and then dripped into hexane (100 times thevolume of CH₂Cl₂). The precipitate was filtered to yield 5.59 (81.5%) ofproduct.

O.N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(2′-isobutyryl-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine(5.58 g) was dissolved in pyridine-MeOH—H₂O (65:30:15, 52 mL) at roomtemperature. The solution was cooled to 0° C. and 52 mL of 2 N NaOH inEtOH-MeOH (95:5) was added slowly, followed by stirring for 2 hours at0° C. Glacial acetic acid was added to pH 6, and saturated NaHCO₃ wasadded to pH 7. The reaction mixture was evaporated under reducedpressure and the residue coevaporated with toluene. The residue was thendissolved in EtOAc (150 mL) and washed 3× with saturated NaHCO₃. Theorganic phase was evaporated and the residue purified by silica gelcolumn chromatography using EtOAc-MeOH (95:5) as the eluent, yielding3.85 g (78.3%) of product.

P.N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine(3.84 g) was dissolved in anhydrous CH₂Cl₂ (79 mL), anhydrous pyridine(5 mL) and DMAP (2.93 g) at room temperature under argon. The solutionwas cooled to 0° C. and trifluoromethanesulfonic anhydride (1.99 mL) wasslowly added with stirring. The reaction mixture was stirred at roomtemperature for 1 hour then poured into 100 mL of saturated NaHCO₃. Theaqueous phase was extracted three times with cold CH₂Cl₂. The organicphase was dried over MgSO₄, evaporated and coevaporated with anhydrousMeCN to yield a crude product.

Q.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-ribofuranosyl)guanine

The crude product from Example 1-P, i.e.N²-isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-arabinofuranosyl)guaninewas dissolved in anhydrous THF (113 mL) under argon at 0° C. 1 M(nBu)₄N⁺F⁻ (dried by coevaporation with pyridine) in THF (36.95 mL) wasadded with stirring. After 1 hour, a further aliquot of (nBu)₄N⁺F⁻ inTHF (36.95 mL) was added. The reaction mixture was stirred at 0° C. for5 hours and stored overnight at −30° C. The reaction mixture wasevaporated under reduced pressure and the residue dissolved in CH₂Cl₂(160 mL) and extracted five times with deionized water. The organicphase was dried over MgSO₄ and evaporated. The residue was purified bysilica gel column chromatography using EtOAc-MeOH (95:5) to yield 5.25 gof product.

R. N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)guanine

N²-isobutyryl-9-(2′-deoxy-2′-fluoro-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-ribofuranosyl)guanine(3.85 g) was dissolved in MeOH (80 mL) at room temperature. Pre-washedDowex 50W resin (12.32 cm³) was added and the reaction mixture stirredat room temperature for 1 hour. The resin was filtered and the filtrateevaporated to dryness. The resin was washed withpyridine-triethylamine-H₂O (1:3:3) until filtrate was clear. Thisfiltrate was evaporated to obtain an oil. The residues from bothfiltrates were combined in H₂O (200 mL) and washed with CH₂Cl₂ (3×100mL). The aqueous phase was evaporated to dryness and the residuerecrystallized from hot MeOH to yield 0.299 g of product as a whitepowder. The remaining MeOH solution was purified by silica gel columnchromatography to further yield 0.783 g of product by elution withEtOH-MeOH (4:1).

S.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4-dimethoxytrityl]-β-D-ribofuranosyl)guanine

N²-isobutyryl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)guanine (1.09 g)was dissolved in pyridine (20 mL) and triethylamine (0.56 mL) at roomtemperature under argon. 4,4′-Dimethoxytrityl chloride (1.20 g, 1.15molar eq.) was added and the reaction mixture stirred at roomtemperature for 5 hours. The mixture was transferred to a separatoryfunnel and extracted with diethyl ether (100 mL). The organic phase waswashed with saturated NaHCO₃ (3×70 mL), and the aqueous phaseback-extracted three times with diethyl ether. The combined organicphases were dried over MgSO₄ and triethylamine (4 mL) was added tomaintain the solution at basic pH. The solvent was evaporated and theresidue purified by silica gel column chromatography usingEtOAc-triethylamine (100:1) and then EtOAc-MeOH-triethylamine (95:5:1)as eluents yielding 1.03 g of product.

T.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4-dimethoxytrityl]-guanosine-3′-O—N,N-diisopropyl-β-D-cyanoethylphosphoramidite

N²-isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4′-dimethoxytrityl])-β-D-ribofuranosyl)guanine(0.587 g) was dissolved in anhydrous CH₂Cl₂ (31 mL) anddiisopropylethylamine (0.4 mL) at room temperature under argon. Thesolution was cooled to 0° C. andchloro(diisopropylamino)-β-cyanoethoxyphosphine (0.42 mL) was slowlyadded. The reaction mixture was allowed to warm to room temperature andstirred for 3.5 hours. CH₂Cl₂-triethylamine (100:1, 35 mL) was added andthe mixture washed with saturated NaHCO₃ (6 mL). The organic phase wasdried over MgSO₄ and evaporated under reduced pressure. The residue waspurified by silica gel column chromatography usinghexane-EtOAc-triethylamine (75:25:1) for 2 column volumes, thenhexane-EtOAc-triethylamine (25:75:1), and finally EtOAc-triethylamine.The product-containing fractions were pooled and the solvent evaporatedunder reduced pressure. The resulting oil was coevaporated twice withMeCN and dried under reduced pressure. The resulting white solid wasdissolved in CH₂Cl₂ (3 mL) and dripped into stirring hexane (300 mL).The resulting precipitate was filtered and dried under reduced pressureto yield 0.673 g (88%) of product.

Example 2 Preparation of 2′-Deoxy-2′-cyano Modified Oligonucleotides A.N⁶-Benzoyl-[2′-deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)

2′-Deoxy-2′-cyanoadenosine is prepared by the free radical replacementof the 2′-iodo group of2′-deoxy-2′-iodo-3′,5′-O-(disiloxytetraisopropyl)-N⁶-benzoyladenosineaccording to a similar procedure described by Parkes and Taylor[Tetrahedron Letters, 29, 2995 (1988)]. 2′-Deoxy-2′-iodoadenosine wasprepared by Ranganathan as described in Tetrahedron Letters, 15, 1291(1977), and disilyated as described by Markiewicz and Wiewiorowski[Nucleic Acid Chemistry, Part 3, pp. 222-231, L. B. Townsend and R. S.Tipson, Eds., J. Wiley and Sons, New York, 1986. This material istreated with hexamethylditin, AIBN, and t-butylisocyanate in toluene toprovide protected 2′-deoxy-2′-cyanoadenosine. This material, afterselective deprotection, is converted to its 5′-DMT-3′-phosphoramidite asdescribed in Example 1A.

B.2′-Deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl)-uridine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)

2′-Deoxy-2′-iodouridine (or 5-methyluridine), 3′,5′-disilylated asdescribed above, is converted to the 2′-iodo derivative bytriphenylphosphonium methyl iodide treatment as described by Parkes andTaylor [Tetrahedron Letters, 29, 2995 (1988)]. Application of freeradical reaction conditions as described by Parkes and Taylor providesthe 2′-cyano group of the protected nucleoside. Deprotection of thismaterial and subsequent conversion to the protected monomer as describedabove provides the requisite phosphoramidite.

C.2′-Deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)

2′-Deoxy-2′-iodocytidine is obtained from the corresponding uridinecompound described above via a conventional keto to amino conversion.

D.2′-Deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl)-guanosine-3′-O—(N,N-diisopropyl-b-cyano-ethylphosphoramidite)

2′-Deoxy-2′-cyanoguanosine is obtained by the displacement of thetriflate group in the 2′-position (arabinosugar) of 3′,5′-disilylatedN²-isobutrylguanosine. Standard deprotection and subsequent reprotectionprovides the title monomer.

Example 3 Preparation of 2′-Deoxy-2′-(trifluoromethyl) ModifiedOligonucleotides

The requisite 2′-deoxy-2′-trifluoromethyribosides of nucleic acid basesA, G, U(T), and C are prepared by modifications of a literatureprocedure described by Chen and Wu [Journal of Chemical Society, PerkinTransactions, 2385 (1989)]. Standard procedures, as described in Example1A, are employed to prepare the 5′-DMT and 3′-phosphoramidites as listedbelow.

A.N⁶-Benzoyl-[2′-deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-di-isopropyl-β-cyanoethylphosphoramidite) B.2′-Deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)uridine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)C.2′-Deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)D.2′-Deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)-guanosine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)Example 4 Preparation of 2′-Deoxy-2′-(trifluoromethoxy) ModifiedOligonucleotides

The requisite 2′-deoxy-2′-O-trifluoromethyribosides of nucleic acidbases A, G, U(T), and C are prepared by modifications of literatureprocedures described by Sproat et al. [Nucleic Acids Research, 18, 41(1990)] and Inoue et al. [Nucleic Acids Research, 15: 131 (1987)].Standard procedures, as described in Example 1A, are employed to preparethe 5′-DMT and 3′-phosphoramidites as listed below.

A.N⁶-Benzoyl-[2′-deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)B.2′-Deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)-uridine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)C.2′-Deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)D.2′-Deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)-guanosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)Example 5 Preparation of 2′-Deoxy-2′-O-alkyl Modified Oligonucleotides

Illustrative 2′-O-alkyl (2′-alkoxy) modified oligonucleotides areprepared from appropriate precursor nucleotides that in turn areprepared starting from a commercial nucleoside. The nucleoside, eitherunblocked or appropriately blocked as necessary to protected exocyclicfunctional groups on their heterobases, are alkylated at the 2′-Oposition. This 2′-O-alkylated nucleosides is converted to a5′-O-dimethoxytrityl protected nucleosides and 3′-O-phosphitylated togive a phosphoramidite. The phosphoramidites are incorporated inoligonucleotides using standard machine cycle solid phasephosphoramidite oligonucleotide chemistry. For illustrative purposes thesynthesis of 2-O-nonyladenosine, 2-O-propyluridine, 2-O-methylcytidine,2′-O-octadecylguanosine,2′-O—[(N-phthalimido)prop-3-yl]-N⁶-benzoyladenosine and2-O-[(imidazol-1-yl)but-4-yl]adenosine are given. Other 2′-O-alkylatednucleosides are prepared in a like manner using an appropriate startingalkyl halide in place of the illustrated alkyl halides. For certain2′-O-aminoalkyl compounds of the invention, protected amines, e.g.phthalimido, were used during alkylation, subsequent tritylation andphosphitylation. After incorporation into the oligonucleotide ofinterest, the 2′-O-protected aminoalkyl moiety are deblocked to yieldthe free amino compound, i.e 2′-O—(CH₂)_(n)—NH₂.

A.N⁶-Benzoyl-[2′-deoxy-2′-O-nonyl-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)2′-O-Nonyladenosine

To a solution of 10 g of adenosine in 400 ml of dimethyl formamide wasadded 2.25 g of 60% sodium hydride (oil). After one hour, 8.5 ml of1-bromononane was added. The reaction was stirred for 16 hours. Ice wasadded and the solution evaporated in vacuo. Water and ethyl acetate wereadded. The organic phase was separated, dried, and evaporated in vacuoto give a white solid, which was recrystallized from ethanol to yield4.8 g of the title compound, m.p. 143-144° C. analysis for: C₁₉H₃₁N₅O₄.Calculated: C, 57.99; H, 7.94; N, 1779. Found: C, 58.13; H, 7.93; N,17.83.

2′-O-Nonyl-N⁶-benzoyladenosine

2′-O-Nonyladenosine was treated with benzoyl chloride in a mannersimilar to the procedure of B. L. Gaffney and R. A. Jones, TetrahedronLett., Vol. 23, p. 2257 (1982). After chromatography on silica gel(ethyl acetate-methanol), the title compound was obtained. Analysis for:C₂₆H₃₅N₅O₅ Calculated: C, 62.75; H, 7.09; N, 17.07. Found: C, 62.73; H,14.07; N, 13.87.

2′-O-Nonyl-5′-O-dimethoxytrityl-N⁶-benzoyladenosine

To a solution of 4.0 g of 2′-O-nonyl-N⁶-benzoyladenosine in 250 ml ofpyridine was added 3.3 g of 4,4′-dimethoxytrityl chloride. The reactionwas stirred for 16 hours. The reaction was added to ice/water/ethylacetate, the organic layer was separated, dried, and concentrated invacuo to a gum. 5.8 g of the title compound was obtained afterchromatography on silica gel (ethyl acetate-methanol triethylamine).Analysis for: C₄₇H₅₃N₅O₇. Calculated: C, 70.56; H, 6.68; N, 8.75. Found:C, 70.26; H, 6.70; N, 8.71.

N⁶-Benzoyl-5′-O-dimethoxytrityl-2′-O-nonyladenosine-3′-O,N,N-diisopropyl-β-cyanoethylPhosphoramidite

2′-O-nonyl-5′-O-dimethoxytrityl-N-benzoyladenosine was treated with(β-cyanoethoxy)chloro(N,N-diisopropyl)aminephosphane in a manner similarto the procedure of F. Seela and A. Kehne, Biochemistry, Vol. 26, p.2233 (1987). After chromatography on silica gel (E=OAC/hexane), thetitle compound was obtained as a white foam.

B.2′-Deoxy-2′-O-propyl-5′-O-(4,4′-dimethoxy-trityl)-uridine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)3′,5′-O-(1,1,3,3)Tetraisopropyl-1,3-disiloxanediyluridine

With stirring, uridine (40 g, 0.164 mol) and1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol)were added to dry pyridine (250 mL). After stirring for 16 h at 25° C.,the reaction was concentrated under reduced pressure to an oil. The oilwas dissolved in methylene chloride (800 mL) and washed with sat'dsodium bicarbonate (200 g) scrub column. The product was recovered byelution with methylene chloride-methanol (97:3). The appropriatefractions were combined, evaporated under reduced pressure and dried at25° C./0.2 mmHg for 1 h to give 65 g (84%) of tan oil; TLC purity 95%(Rf 0.53, ethyl acetate-methanol 95:5); PMR (CDCl₃) δ 7.87 (d, 1, H-6),5.76 (d, 1, H-5), 5.81 (s, 1, H-1′).

N³-(4-Toluoyl)-3′-5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxane-diyluridine

4-Toluoyl chloride (19.6 g, 0.127 mol) was added over 30 min to astirred solution of3′,5′-O-(1,1,3,3)-tetra-isopropyl-1,3-disiloxanediyluridine (56 g, 0.115mol) and triethylamine (15.1 g, 0.15 mol) in dimethylacetamide (400 mL)at 5° C. The mixture was allowed to warm to 25° C. for 3 h and thenpoured onto ice water (3.5 L) with stirring. The resulting solid wascollected, washed with ice water (3×500 mL) and dried at 45° C./0.2 mmHgfor 5 h to afford 49 g (70%) of tan solid; mp slowly softens above 45°C.; TLC purity ca. 95% (Rf 0.25, hexanes-ethyl acetate 4:1); PMR (DMSO)δ 7.9 (H-6), 7.9-7.4 (Bz), 5.8 (H-5), 5.65 (HO-2′), 5.6 (H-1′), 2.4(CH₃—Ar).

N³-(4-Toluoyl)-2′-O-propyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediyluridine

A mixture ofN³-(4-toluoyl)-3′-5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxane-diyluridine(88 g, 0.146 mol, 95% purity), silver oxide (88 g, 0.38 mol) and toluene(225 mL) was evaporated under reduced pressure. More toluene (350 mL)was added and an additional amount (100 mL) was evaporated. Under anitrogen atmosphere, propyl iodide was added in one portion and thereaction was stirred at 40° C. for 16 h. The silver salts were collectedand washed with ethyl acetate (3×150 mL). The combined filtrate wasconcentrated under reduced pressure. The residue was dissolved in aminimum of hexanes, applied on a silica gel column (800 g) and elutedwith hexanes-ethyl acetate (9:1→4:1). The appropriate fractions werecombined, concentrated under reduced pressure and dried at 25° C./0.2mmHg for 1 h to provide 68 g (74%) of tan oil; TLC purity 95% (Rf 0.38,hexanes-ethyl acetate 4:1); PMR (CDCl₃) δ 8.1-7.3 (m, 6, H-6 and Bz),5.8 (H-5), 5.76 (H-1′).

2′-O-Propyluridine

A solution ofN³-(4-toluoyl)-2′-O-propyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediyluridine(27 g) in methanol (400 mL) and ammonium hydroxide (50 mL) was stirredfor 16 h at 25° C. The reaction was concentrated under reduced pressureto an oil; TLC homogenous (Rf 0.45, ethyl acetate-methanol 95:5).

The oil was dissolved in toluene (100 mL) and the solution wasevaporated under reduced pressure to dryness. The residue was dissolvedin tetrahydrofuran (300 mL). Tetrabutylammonium fluoride solution (86mL, 1 M in tetrahydrofuran) was added and the reaction was stirred at25° C. for 16 h. The pH was adjusted to 7 with Amberlite IRC-50 resin.The mixture was filtered and the resin was washed with hot methanol(2×200 mL). To the combined filtrate was added silica gel (40 g). Thesuspension was concentrated under reduced pressure to a dry powder. Theresidue was placed on top of a silica gel column (500 g) and eluted withethyl acetate and then ethyl acetate-methanol (9:1). The appropriatefractions were combined, evaporated under reduced pressure and dried at90° C./0.2 mmHg for 5 h to yield 8.0 g (70%) of light tan solid; TLCpurity 98% (Rf 0.45, ethyl acetate-methanol 4:1); PMR (DMSO) δ 11.37(H—N³), 7.9 (H-6), 5.86 (H-1′), 5.65 (H-5), 5.2 (HO-3′,5′).

5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-propyluridine

2′-O-Methyluridine (8.0 g) was evaporated under reduced pressure to anoil with pyridine (100 mL). To the residue was added4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl, 11.5 g, 0.34 mol) andpyridine (100 mL). The mixture was stirred at 25° C. for 1.5 h and thenquenched by the addition of methanol (10 mL) for 30 min. The mixture wasconcentrated under reduced pressure and the residue was chromatographedon silica gel (250 g, hexanes-ethyl acetate-triethylamine 80:20:1 andthen ethyl acetate-triethylamine 99:1). The appropriate fractions werecombined, evaporated under reduced pressure and dried at 25° C./0.2 mmHgfor 1 h to provide 17.4 g (100%, 30% from uridine) of tan foam; TLCpurity 98% (Rf 0.23, hexanes-ethyl acetate 4:1); PMR (DMSO) δ 11.4(H—N³), 7.78 (H-6), 7.6-6.8 (Bz), 5.8 (H-1′), 5.3 (H-5′), 5.25 (HO-3′),3.7 (CH₃O-Bz).

[5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-propyluridin-3′-O-yl]-N,N-diisopropylaminocyanoethoxyphosphoramidite

The product was prepared in the same manner as the adenosine analogabove by starting with intermediate5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-propyluridine and using ethylacetate-hexanes-triethylamine 59:40:1 as the chromatography eluent togive the product as a solid foam in 60% yield (18% from uridine); TLChomogenous diastereomers (Rf 0.58; 0.44, ethylacetate-hexanes-triethylamine 59:40:1); ³¹P-NMR (CDCl₃, H₃PO₄ std.) δ148.11; 148.61 (diastereomers).

C.2′-Deoxy-2′-O-methyl-5′-O-(4,4′-dimethoxy-trityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)

Two methods will be described for the preparation of the intermediateN⁴-benzoyl-2′-O-methylcytidine. Method A involves blocking of the 3′-5′sites with the TIPS-Cl reagent to allow alkylation only on the 2′position. Method B uses a direct alkylation of cytidine followed byseparation of the resulting mixture. The overall yields are comparable.

Method A: 3′,5′-O-(1,1,3,3)-Tetraisopropyl-1,3-disiloxanediylcytidine

With stirring, cytidine (40 g, 0.165 mol) and1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol)were added to dry pyridine (250 mL). After stirring for 16 h at 25° C.,the reaction was concentrated under reduced pressure to an oil. The oilwas dissolved in methylene chloride (800 mL) and washed with sat'dsodium bicarbonate (2×300 mL). The organic layer was passed through asilica gel (200 g) scrub column. The product was recovered by elutionwith methylene chloride:methanol (97:3). The appropriate fractions werecombined, evaporated under reduced pressure and dried at 25° C./0.2 mmHgfor 1 h to give 59.3 g (77%) of oil (the product may be crystallizedfrom ethyl acetate as white crystals, mp 242-244° C.); TLC purity 95%(Rf 0.59, ethyl acetate-methanol 9:1); PMR (DMSO) δ 7.7 (H-6), 5.68(H-5), 5.61 (HO-2′), 5.55 (H-1′).

N⁴-(Benzoyl)-3′-5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediylcytidine

Benzoyl chloride (18.5 g, 0.13 mol) was added over 30 min to a stirredsolution of 3′,5′-O-(1,1,3,3)-tetraisopropyl-1,3-disiloxanediylcytidine(58 g, 0.12 mol) and triethylamine (15.6 g, 0.16 mol) indimethylacetamide (400 mL) at 5° C. The mixture was allowed to warm to25° C. for 16 h and then poured onto ice water (3.5 L) with stirring.The resulting solid was collected, washed with ice water (3×500 mL) anddried at 45° C./0.2 mmHg for 5 h to provide 77 g (100%) of solid; TLCpurity ca. 90% (Rf 0.63, chloroform-methanol 9:1); PMR (CDCL₃) δ 8.32(H-6). Lit. mp 100-101° C.

N⁴-(Benzoyl)-2′-O-methyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediylcytidine

A mixture ofN⁴-(benzoyl)-3′-5′-O-(1,1,3,3)tetra-isopropyl-1,3-disiloxanediylcytidine(166 g, 0.25 mol, 90% purity), silver oxide (150 g, 0.65 mol) andtoluene (300 mL) was evaporated under reduced pressure. More toluene(500 mL) was added and an additional amount (100 mL) was evaporated.Under a nitrogen atmosphere, methyl iodide was added in one portion andthe reaction was stirred at 40_(i)C for 16 h. The silver salts werecollected and washed with ethyl acetate (3×150 mL). The combinedfiltrate was concentrated under reduced pressure. The residue wasdissolved in a minimum of methylene chloride, applied to a silica gelcolumn (1 kg) and eluted with hexanes-ethyl acetate (3:2®1:1). Theappropriate fractions were combined, concentrated under reduced pressureand dried at 45° C./0.2 mmHg for 1 h to yield 111 g (66%) of oil; TLCpurity ca. 90% (Rf 0.59, hexanes-ethyl acetate 3:2). PMR (CDCl₃) δ 8.8(br s, 1, H—N⁴), 8.40 (d, 1, H-6), 8.0-7.4 (m, 6, H-5 and Bz), 5.86 (s,1, H-1), 3.74 (s, 3, CH₃O-2′).

N⁴-Benzoyl-2′-O-methylcytidine

A solution ofN⁴-(benzoyl)-2′-O-methyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediylcytidine(111 g, 0.18 mol) in methanol (160 mL) and tetrahydrofuran (640 mL) wastreated with tetrabutylammonium fluoride solution (368 mL, 1 M intetrahydrofuran). The reaction was stirred at 25° C. for 16 h. The pHwas adjusted to 7 with Amberlite IRC-50 resin. The mixture was filteredand the resin was washed with hot methanol (2×200 mL). The combinedfiltrate was concentrated under reduced pressure, absorbed on silica gel(175 g) and chromatographed on silica gel (500 g, ethyl acetate-methanol19:1®4:1). Selected fractions were combined, concentrated under reducedpressure and dried at 40° C./0.2 mmHg for 2 h to yield 28 g (42.4%,21.5% from cytidine) of solid; TLC homogenous (Rf 0.37, ethyl acetate).mp 178-180° C. (recryst. from ethanol); PMR (CDCl₃) δ 11.22 (br s, 1,H—N⁴), 8.55 (d, 1, H-6), 8.1-7.2 (m, 6, H-5 and Bz), 5.89 (d, 1, H-1′),5.2 (m, 2, HO-3′,5′), 3.48 (s, 3, CH₃O-2′).

Method B: N⁴-Benzoyl-2′-O-methylcytidine

Cytidine (100 g, 0.41 mol) was dissolved in warm dimethylformamide (65°C., 1125 mL). The solution was cooled with stirring to 0° C. A slow,steady stream of nitrogen gas was delivered throughout the reaction.Sodium hydride (60% in oil, washed thrice with hexanes, 18 g, 0.45 mol)was added and the mixture was stirred at 0_(i)C for 45 min. A solutionof methyl iodide (92.25 g, 40.5 mL, 0.65 mol) in dimethylformamide (400mL) was added in portions over 4 h at 0° C. The mixture was stirred for7 h at 25° C. and then filtered. The filtrate was concentrated todryness under reduced pressure followed by coevaporation with methanol(2×200 mL). The residue was dissolved in methanol (350 mL). The solutionwas adsorbed on silica gel (175 g) and evaporated to dryness. Themixture was slurried in dichloromethane (500 mL) and applied on top of asilica gel column (1 kg). The column was eluted with a gradient ofdichloromethane-methanol (10:1®2:1). The less polar 2′,3′-dimethyl sideproduct was removed and the coeluting 2′ and 3′-O-methyl productcontaining fractions were combined and evaporated under reduced pressureto a syrup. The syrup was dissolved in a minimum of hot ethanol (ca. 150mL) and allowed to cool to 25° C. The resulting precipitate (2′ lesssoluble) was collected, washed with ethanol (2×25 ml) and dried to give15.2 g of pure 2′-O-methylcytidine; mp 252-254° C. (lit. mp 252-254°C.); TLC homogenous (Rf 0.50, dichloromethane-methanol 3:1, (Rf of 3′isomer is 0.50 and the dimethyl product is 0.80). The filtrate wasevaporated to give 18 g of a mixture of isomers and sodium iodide.

The pure 2′-O-methylcytidine (15.2 g, 0.060 mol) was dissolved in asolution of benzoic anhydride (14.7 g, 0.12 mol) in dimethylformamide(200 mL). The solution was stirred at 25° C. for 48 h and thenevaporated to dryness under reduced pressure. The residue was trituratedwith methanol (2×200 mL), collected and then triturated with warm ether(300 mL) for 10 min. The solid was collected and triturated with hot2-propanol (50 mL) and allowed to stand at 4° C. for 16 h. The solid wascollected and dried to give 17 g of product. The crude filtrate residue(18 g) of 2′-O-methylcytidine was treated with benzoic anhydride (17.3g, 0.076 mol) in dimethylformamide (250 mL) as above and triturated in asimilar fashion to give an additional 6.7 g of pure product for a totalyield of 23.7 g (16% from cytidine) of solid; TLC homogenous (Rf 0.25,chloroform-methanol 5:1, cospots with material produced from the otherroute).

N⁴-Benzoyl-5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-methylcytidine

N⁴-Benzoyl-2′-O-methylcytidine (28 g, 0.077 mol) was evaporated underreduced pressure to an oil with pyridine (400 mL). To the residue wasadded 4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl, 28.8 g, 0.085 mol)and pyridine (400 mL). The mixture was stirred at 25° C. for 2 h andthen quenched by the addition of methanol (10 mL) for 30 min. Themixture was concentrated under reduced pressure and the residue waschromatographed on silica gel (500 g, hexanes-ethylacetate-triethylamine 60:40:1 and then ethyl acetate-triethylamine99:1). The appropriate fractions were combined, evaporated under reducedpressure and dried at 40° C./0.2 mmHg for 2 h to give 26 g (74%, 16%from cytidine) of foam; TLC homogenous (Rf 0.45, ethyl acetate); PMR(DMSO) δ 11.3 (H—N⁴), 8.4-6.9 (H-6, H-5, Bz), 5.95 (H-1′), 5.2 (HO-3′),3.7 (s, 6, CH₃O-trit.), 3.5 (s, 3, CH₃O-2′)

[N⁴-Benzoyl-5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-methylcytidin-3′-O-yl]-N,N-diisopropylamino-cyano-ethoxyphosphoramidite

The product was prepared in the same manner as the adenosine analogabove by starting with intermediateN⁴-benzoyl-5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-methylcytidine andusing ethyl acetate-hexanes-triethylamine 59:40:1 as the chromatographyeluent to give the product as a solid foam in 71% yield (11% fromcytidine); TLC homogenous diastereomers (Rf 0.46; 0.33, ethylacetate-hexanes-triethylamine 59:40:1); ³¹P-NMR (CD₃CN, H₃PO₄ std.) δ150.34; 151.02 (diastereomers).

D.21-Deoxy-2′-octadecyl-5′-O-(4,4′-dimethoxy-trityl)-guanosine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)2,6-Diamino-9-(2-O-octadecyl-β-D-ribofuranosyl)purine

2,6-Diamino-9-(β-D-ribofuranosyl)purine (50 g, 180 mmol) and sodiumhydride (7 g) in DMF (1 L) were heated to boiling for 2 hr.Iodooctadecane (100 g) was added at 150° C. and the reaction mixtureallowed to cool to RT. The reaction mixture was stirred for 11 days atRT. The solvent was evaporated and the residue purified by silica gelchromatography. The product was eluted with 5% MeOH/CH₂Cl₂. Theappropriate fractions were evaporated to yield the product (11 g). ¹HNMR (DMSO-d₆) δ 0.84 (t, 3, CH₂), 1.22 (m, 32, O—CH₂—CH₂—(CH₂)₁₆), 1.86(m, 2, O—CH₂CH₂), 3.25 (m, 2, O—CH₂), 3.93 (d, 1, 4′H), 4.25 (m, 1,3′H), 4.38 (t, 1, 2′H), 5.08 (d, 1,3′-OH), 5.48 (t, 1,5′-OH), 5.75 (s,2, 6-NH₂), 5.84 (d, 1,1′-H), 6.8 (s, 2, 2-NH₂), and 7.95 (s, 1,8-H).

2′-O-Octadecylguanosine

2,6-Diamino-9-(2-O-octadecyl-β-D-ribofuranosyl)purine (10 g) in 0.1 Msodium phosphate buffer (50 ml, pH 7.4), 0.1 M tris buffer (1000 ml, pH7.4) and DMSO (1000 ml) was treated with adenosine deaminase (1.5 g) atRT. At day 3, day 5 and day 7 an additional aliquot (500 mg, 880 mg and200 mg, respectively) of adenosine deaminase was added. The reaction wasstirred for a total of 9 day and purification by silica gelchromatography yielded the product (2 g). An analytical sample wasrecrystallized from MeOH. ¹H NMR (DMSO-d₆) δ 0.84 (t, 3, CH₃), 1.22 (s,32, O—CH₂—CH₂—(CH₂)₁₆), 5.07 (m, 2,3′-OH and 5′-OH), 5.78 (d, 1, 1′H),6.43 (s, 2, NH₂), 7.97 (s, 1, 8H) and 10.64 (s, 1, NH₂). Anal. Calcd.for C₂₈H₄₉N₅O₅: C, 62.80; H, 9.16; N, 12.95. Found: C, 62.54; H, 9.18;N, 12.95.

N²-Isobutyryl-2′-O-octadecylguanosine

2′-O-Octadecylguanosine (1.9 g) in pyridine (150 ml) was cooled in anice bath, and treated with trimethylsilyl chloride (2 g, 5 eq) andisobutyryl chloride (2 g, 5 eq). The reaction mixture was stirred for 4hours, during which time it was allowed to warm to room temperature. Thesolution was cooled, water added (10 mL) and stirred for an additional30 minutes. Concentrated ammonium hydroxide (10 mL) was added and thesolution concentrated in vacuo. The residue was purified by silica gelchromatography (eluted with 3% MeOH/EtOAc) to yield 1.2 g of product. ¹HNMR (DMSO-d₆) δ 0.85 (t, 3, CH₃), 1.15 (m, 38, O—CH₂CH₂(CH₂)₁₆ andCH(CH₃)₂), 2.77 (m, 1, CH(CH₃)₂), 4.25 (m, 2, 2′H, 3′H), 5.08 (t,1,5′-OH), 5.12 (d, 1,3′-OH), 5.87 (d, 1,1′-H), 8.27 (s, 1,8-H), 11.68(s, 1, NH₂) and 12.08 (s, 1, NH₂). Anal. Calcd. for C₃₂H₅₅N₅O₆: C,63.47; H, 9.09; N, 11.57. Found: C, 63.53; H, 9.20; N, 11.52.

N²-Isobutyryl-5′-dimethoxytrityl-2′-O-octadecylguanosine

N²-Isobutyryl-2′-O-octadecylguanosine was converted to theN²-isobutyryl-5′-dimethoxytrityl-2′-O-octadecylguanosine as per theprocedure for adenosine above.

[N²-Isobutyryl-5′-dimethoxytrityl-2′-O-octadecylguan-3′-O-yl]-N,N-diisopropylamino-cyanoethoxyphosphoramidite

The product was prepared in the same manner as the adenosine analogabove by starting with intermediateN²-iso-butyryl-5′-dimethoxytrityl-2′-O-octadecylguanosine.

E.N⁶-Benzoyl-2′-[N-phthalimido)prop-3-yl]5′-O(4,4′-dimethoxytrityl)]adenosine3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)2′-O—[(N-phthalimido)prop-3-yl]adenosine

The title compound was prepared as per the 2′-O-nonyladenosine procedureusing N-(3-bromopropyl)phthalimide. Chromatography on silica gel give awhite solid, m.p. 123-124° C. Analysis for: C₂₁H₂₂N₆O₆. Calculated: C,55.03; H, 4.88; N, 18.49. Found: C, 55.38; H, 4.85; N, 18.46.

2′-O—[(N-phthalimido)prop-3-yl]-N⁶-benzoyladenosine

Benzoylation of 2′-O—[(N-phthalimido)prop-3-yl]-adenosine as per the2′-O-nonyladenosine procedure above give the title compound. Analysisfor: C₂₈H₂₆N₆O₇. Calculated: C, 60.21; H, 4.69; N, 15.05. Found: C,59.94; H, 4.66; N, 14.76.

2′-O—[(N-phthalimido)prop-3-yl]-5′-O-dimethoxytrityl-N⁶-benzoyl-adenosine

The title compound was prepared from2′-O—[(N-phthalimido)prop-3-yl]-N⁶-benzoyladenosine as per the2′-O-nonyladenosine above. Analysis for: C₄₉H₄₄N₆O₉. Calculated: C,68.36; H. 5.15; N, 9.76. Found: C, 68.16; H, 5.03; N, 9.43.

N₆-benzoyl-5′-O-dimethoxytrityl-2′-O—[(N-phthalimido)prop-3-yl]-adenosine-3′-O,N,N-diisopropyl-β-cyanoethylphosphoramidite

The title compound was prepared from2′-O—[(N-phthalimido)prop-3-yl]-5′-O-dimethoxytrityl-N⁶-benzoyladenosineas above for the 2′-O-nonyladenosine compound. A white foam wasobtained.

F.N⁶-Benzoyl-2′-[(imidazol-1-yl)butyl-4-yl]5′O(4,4′di-methoxytrityl)]adenosine-3′-O(N,N-diisopropyl-β-cyanoethylphosphoramidite)2′-O-[imidizo-1-yl-(but-4-yl)]adenosine

The title compound can be prepared as per the 2′-O-nonyladenosineprocedure using 1-(4-bromobutyl)imidazole in place of 1-bromononane.

2′-O-[(imidizol-1-yl)but-4-yl]-N⁶-benzoyladenosine

Benoylation of 2′-O-[(imidizol-1-yl)but-4-yl)]-adenosine as per the2′-O-nonyladenosine procedure above will give the title compound.

2′-O-[(imidizol-1-yl)but-4-yl]-5′-O-dimethoxytrityl-N⁶-benzoyladenosine

The title compound can be prepared from2′-O-[(imidizol-1-yl)but-4-yl]adenosine as per the 2′-O-nonyladenosineprocedure above.

N⁶-benzoyl-5′-O-dimethoxytrityl-2′-O-[(imidizol-1-yl)but-4-yl]-adenosine-3′-O,N,N-diisopropyl-β-cyanoethylphosphoramidite

The title compound can be prepared from2′-O-[(imidizol-1-yl)but-4-yl)]-5′-O-dimethoxytrityl-N⁶-benzoyladenosineas per the 2′-O-nonyladenosine procedure above.

Example 6 Preparation of 2′-Deoxy-2′-(vinyloxy) ModifiedOligonucleotides

The requisite 2′-deoxy-2′-O-vinyl ribosides of nucleic acid bases A, G,U(T), and C are prepared by modifications of literature proceduresdescribed by Sproat et al. [Nucleic Acids Research, 18, 41 (1990)] andInoue et al. [Nucleic Acids Research, 15, 6131 (1987)]. In this case1,2-dibromoethane is coupled to the 2′-hydroxyl and subsequentdehydrobromination affords the desired blocked 2′-vinyl nucleoside.Standard procedures, as described in Example 1A, are employed to preparethe 5′-DMT and 3′-phosphoramidites as listed below.

A.N⁶-Benzoyl-[2′-deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)B.2′-Deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxy-trityl)-uridine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)C.2′-Deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxy-trityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)D.2′-Deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxy-trityl)-guanosine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)Example 7 Preparation of 2′-Deoxy-2′-(allyloxy) ModifiedOligonucleotides

The requisite 2′-deoxy-2′-O-allyl ribosides of nucleic acid bases A, G,U(T), and C are prepared by modifications of literature proceduresdescribed by Sproat et al. [Nucleic Acids Research, 18, 41 (1990)] andInoue et al. [Nucleic Acids Research, 15, 6131 (1987)]. Standardprocedures, as described in Example 1A, are employed to prepare the5′-DMT and 3′-phosphoramidites as listed below.

A.N⁶-Benzoyl-[2′-deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)B.2′-Deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxy-trityl)-uridine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)C.2′-Deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxy-trityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)D.2′-Deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxy-trityl)-guanosine-3′-O—(N,N-diisopropyl-β-cyano-ethylphosphoramidite)Example 8 Preparation of 2′-deoxy-2′-(methylthio), (methylsulfinyl) and(methylsulfonyl) Modified Oligonucleotides A.2′-Deoxy-2′-methylthiouridine

2,2′Anhydrouridine (15.5 g, 68.2 mmol) [Rao and Reese, J. Chem. Soc.,Chem. Commun., 997], methanethiol (15.7 g, 327 mmol),1,1,3,3-tetramethylguanidine (39.2 g, 341 mmol) and DMF (150 mL) wereheated at 60° C. After 12 hours, the reaction mixture was cooled andconcentrated under reduced pressure. The residual oil was purified byflash column chromatography on silica gel (300 g). Concentration of theappropriate fractions, which were eluted with CH₂Cl₂-MeOH (9:1), anddrying the residue under reduced pressure gave2′-deoxy-2′-methylthiouridine as a pale yellow solid (14.11 g, 75.4%).Attempts to crystallize the solids from EtOH-hexanes [as reported byImazawa et al., Chem. Pharm. Bull., 23, 604 (1975)] failed and thematerial turned into a hygroscopic foam.

¹H NMR (DMSO-d₆) δ 2.0 (3H, s, SCH₃), 3.34 (1H, dd, J_(3′,2′)=5.4 Hz, 2′H), 3.59 (2H, br m, 5′CH₂), 3.84 (1H, m, 4′ H), 4.2 (1H, dd,J_(3′,4′)=2.2 Hz, 3′ H), 5.15 (1H, t, 5′ OH), 5.62 (1H, t, 3′OH), 5.64(1H, d, J_(C6,C5)=8.2 Hz), 6.02 (1H, d, J_(1′,2′)=6 Hz, 1′ H), 7.82 (1H,d, J_(C5,C6)=8.2 Hz, C6H), 11.38 (1H, br s, NH).

B. 2,2′-Anhydro-5-methyluridine

A mixture of 5-methyluridine (16.77 g, 69.2 mmol), diphenyl carbonate(17.8 g, 83.1 mmol) and NaHCO₃ (100 mg) in hexamethylphosphoramide (175mL) was heated to 150° C. with stirring until evolution of CO₂ ceased(approximately 1 hour). The reaction mixture was cooled and then pouredinto diethyl ether (1 L) while stirring to furnish a brown gum. Repeatedwashings with diethyl ether (4×250 mL) furnished a straw-coloredhygroscopic powder. The solid was purified by short columnchromatography on silica gel (400 g). Pooling and concentratingappropriate fraction, which were eluted with CH₂Cl₂-MeOH (85:15),furnished the title compound as a straw-colored solid (12 g, 77.3%),which crystallized from EtOH as long needles, m.p. 226-227° C.

C. 2′-Deoxy-2′-methylthio-5-methyluridine

2,2′-Anhydro-5-methyluridine (17.02 g, 70.6 mmol), methanethiol (16.3 g,339 mmol), 1,1,3,3-tetramethylguanidine (40.6 g, 353 mmol) and DMF (150mL) were heated at 60° C. After 12 hours, the products were cooled andconcentrated under reduced pressure. The residual oil was purified byshort silica gel column chromatography (300 g). Pooling andconcentrating appropriate fractions, which were eluted with CH₂Cl₂-MeOH(93:7), furnished the title compound as a white foam (15.08 g, 74.1%),which was crystallized from EtOH—CH₂Cl₂ as white needles.

D. 2′-Deoxy-2′-methylsulfinyluridine

To a stirred solution of 2′-deoxy-2′-methylthiouridine (1 g, 3.65 mmol)in EtOH (50 mL) was added a solution of m-chloroperbenzoic acid (50%,1.26 g, 3.65 mmol) in EtOH (50 mL) over a period of 45 minutes at 0° C.the solvent was removed under reduced pressure and the residue purifiedby short silica gel (30 g) column chromatography. Concentration of theappropriate fractions, which were eluted with CH₂Cl₂-MeOH (75:25),afforded the title compound as a white solid (0.65 g, 61.4%).Crystallization from EtOH furnished white granules, m.p. 219-221° C.

¹H NMR (DMSO-d₆) δ 2.5 (3H, s, SOCH₃), 3.56 (2H, br s, 5′CH₂), 3.8 (1H,m, 4′ H), 3.91 (1H, m, 2′ H), 4.57 (1H, m, 3′ H), 5.2 (1H, br s, 5′ OH),5.75 (1H, d, C₅H), 6.19 (1H, d, 3′ OH), 6.35 (1H, d, 1′ H), 7.88 (1H, d,C₆H), 11.43 (1H, br s, NH)

E. 2′-Deoxy-2′-methylsulfonyluridine

To a stirred solution of 2′-deoxy-2′-methyluridine (1 g, 3.65 mmol) inEtOH (50 mL) was added a solution of m-chloroperbenzoic acid (50%, 3.27g, 14.6 mmol) in one portion at room temperature. After 2 hours, thesolution was filtered to separate the white precipitate which wasformed, which upon washing (2×20 mL EtOH and 2×20 mL diethyl ether) anddrying, furnished the title compound as a fine powder (0.76 g, 68%),m.p. 227-228° C.

¹H NMR (DMSO-d₆) δ 3.1 (3H, S, SO₂CH₃), 3.58 (2H, m, 5′ CH₂), 3.95 (1H,m, 2′ H), 3.98 (1H, m, 4′ H), 4.5 (1H, br s, 3′ H), 5.2 (1H, br s, 5′OH), 5.75 (1H, d, C₅H), 6.25 (1H, d, 3′ OH), 6.5 (1H, d, 1′ H), 7.8 (1H,d, C6H), 11.45 (1H, br s, NH).

F. 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiouridine

To a stirred solution of 2′-deoxy-2′-methylthiouridine (1.09 g, 4 mmol)in dry pyridine (10 mL) was added 4,4′-dimethoxytritylchloride (1.69 g,5 mmol) and 4-dimethylaminopyridine (50 mg) at room temperature. Thesolution was stirred for 12 hours and the reaction mixture quenched byadding MeOH (1 mL). The reaction mixture was concentrated under reducedpressure and the residue was dissolved in CH₂Cl₂ (100 mL), washed withsaturated aqueous NaHCO₃ (2×50 mL) and saturated aqueous NaCl (2×50 mL),and dried with MgSO₄. The solution was concentrated under reducedpressure and the residue purified by silica gel (30 g) columnchromatography. Elution with CH₂Cl₂-MeOH-triethylamine (89:1:1)furnished the title compound as a homogenous material. Pooling andconcentrating the appropriate fractions furnished the 5′-O-DMTnucleoside as a foam (1.5 g, 66.5%).

¹H NMR (DMSO-d₆) δ 2.02 (3H, s, SCH₃), 3.15-3.55 (1H, m, 2′CH), 3.75(6H, s, 2 OCH₃), 3.97 (1H, m, 4′ H), 4.24 (1H, m, 3′H), 5.48 (1H, d,C₅H), 5.73 (1H, d, 3′OH), 6.03 (1H, d, C1′H), 6.82-7.4 (13H, m, ArH),6.65 (1H, d, C6H), 11.4 (1H, br s, NH).

G.2′-Deoxy-3′-O—[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiouridine

To a stirred solution of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiouridine (1.5 g, 2.67mmol) in dry THF (25 mL) was added diisopropylethylamine (1.4 mL, 8mmol) and the solution was cooled to 0° C.N,N-Diisopropyl-β-cyanoethylphosphoramidic chloride (1.26 mL, 5.34 mmol)was added dropwise over a period of 15 minutes. The reaction mixture wasthen stirred at room temperature for 2 hours. Ethyl acetate (100 mL,containing 1% triethylamine) was added and the solution washed withsaturated NaCl (2×50 mL) and the organic layer dried over MgSO₄. Thesolvent was removed under reduced pressure and the residue purified byshort silica gel (30 g) column chromatography. Elution withCH₂Cl₂-MeOH-triethylamine (98:1:1) furnished the product as a mixture ofdiastereomers. Evaporation of the appropriate fractions provided thetitle compound as a foam (1.32 g, 64.7%).

¹H NMR (CDCl₃) δ 2.0 and 2.02 (3H, s, SCH₃), 5.3 and 5.35 (1H, 2d, C₅H),6.23 (1H, d, 1′ H), 7.8 and 7.88 (1H, 2d, C₆H) and other protons.

³¹P NMR (CDCl₃) δ 151.68 and 152.2 ppm.

H. 2′-Deoxy-3′,5′-di-O-acetyl-2′-methylthiouridine

2′-Deoxy-2′-methylthiouridine (5.0 g, 18.24 mmol) and acetic anhydride(5.6 mL, 54.74 mmol) were stirred in dry pyridine (30 mL) at roomtemperature for 12 hours. The products were then concentrated underreduced pressure and the residue obtained was purified by short silicagel column chromatography. The appropriate fractions, which were elutedwith CH₂Cl₂-MeOH (9:1), were combined, evaporated under reduced pressureand the residue crystallized from EtOH to give the title compound (6.0g, 91.8%) as white needles, m.p. 132° C.

¹H NMR (CDCl₃) δ 2.17 (3H, s, SCH₃), 2.20 (6H, s, 2 COCH₃), 3.40 (1H, t,2′H), 4.31-4.40 (3H, m, 4′,5′H), 5.31 (1H, m, 3′H), 5.80 (1H, d, C₅H),6.11 (1H, d, 1′ H), 7.45 (1H, d, C₆H), 8.7 (1H, br s, NH).

I. 2′-Deoxy-3′,5′-di-O-acetyl-4-(1,2,4-triazol-yl)-2′-methylthiouridine

Triethylamine (8.4 mL, 60.3 mmol) and phosphoryl chloride (1.2 mL, 12.9mmol) were added to a stirred solution of2′-deoxy-3′,5′-di-O-acetyl-2′-methylthiouridine (4.6 g, 13 mmol) in MeCN(50 mL). 1,2,4-Triazole (4.14 g, 59.9 mmol) was then added and thereactants were stirred at room temperature. After 16 hours,triethylamine-H₂O (6:1, 20 mL) was added, followed by saturated aqueousNaHCO₃ (100 mL), and the resulting mixture was extracted with CH₂Cl₂(2×100 mL). The organic layer was dried with MgSO₄ and evaporated underreduced pressure. The residue was purified by short silica gel columnchromatography. The appropriate fractions, which were eluted withCH₂Cl₂-MeOH (9:1), were evaporated under reduced pressure and theresidue was crystallized from EtOH to give the title compound (3.01 g,56.4%) as needles, m.p. 127-130° C.

¹H NMR (CDCl₃) δ 2.18 (6H, s, 2 COCH₃), 2.30 (3H, s, SCH₃), 3.67 (1H, m,2′H), 4.38-4.50 (3H, m, 4′,5′H), 5.17 (1H, t, 3′H), 6.21 (1H, d, 1′H),7.08 (1H, d, C₅H), 8.16 (1H, s, CH), 8.33 (1H, d, C₆H), 9.25 (1H, s,NH).

J. 2′-Deoxy-2′-methylthiocytidine

2′-Deoxy-3′,5′-di-O-acetyl-4-(1,2,4-triazol-1-yl)-2′-methylthiouridine(3.0 g, 7.5 mmol) was dissolved in a saturated solution of ammonia inMeOH (70 mL) and the solution was stirred at room temperature in apressure bottle for 3 days. The products were then concentrated underreduced pressure and the residue was crystallized from EtOH—CH₂Cl₂ togive the title compound (1.06 g, 51.7%) as crystals, m.p. 201° C.

¹H NMR (DMSO-d₆) δ 1.95 (3H, s, SCH₃), 3.36 (1H, m, 2′H), 3.55 (2H, m,5′CH₂), 3.82 (1H, m, 4′ H), 4.18 (1H, dd, 3′H), 5.75 (1H, d, C₅H), 6.1(1H, d, 1′H), 7.77 (1H, d, C₆H)

Anal calcd. for C₁₀H₁₅N₃O₄S: C, 43.94; H, 5.53; N, 15.37; S, 11.73.Found: C, 44.07; H, 5.45; N, 15.47; S, 11.80.

K. 2′-Deoxy-N⁴-benzoyl-2′-methylthiocytidine

To a stirred solution of 2′-deoxy-2′-methylthiocytidine (0.86 g, 3.15mmol) in dry pyridine (20 mL) was added trimethylchlorosilane (2 mL,15.75 mmol), and stirring continued for 15 minutes. Benzoyl chloride(2.18 mL, 18.9 mmol) was added to the solution followed by stirring for2 hours. The mixture was then cooled in an ice bath and MeOH (10 mL) wasadded. After 5 minutes, ammonium hydroxide (30% aq., 20 mL) was addedand the mixture stirred for 30 minutes. The reaction mixture was thenconcentrated under reduced pressure and the residue purified by shortsilica gel (70 g) column chromatography. Elution with CH₂Cl₂-MeOH (9:1),pooling of the appropriate fractions and evaporation furnished the titlecompound (0.55 g, 46.6%), which crystallized from EtOH as needles, m.p.193-194° C.

L.N⁴-Benzoylamino-1-[2′-deoxy-5′-(4,4′-dimethoxytrityl)-2-methylthio-β-D-ribofuranosyl]pyrimidin-3(2H)-oneor 2′-deoxy-N⁴-benzoyl-5′-(4,4′-dimethoxytrityl)-2′-methylthiocytidine)

To a stirred solution of 2′-deoxy-N⁴-benzoyl-2′-methylthiocytidine (0.80g, 2.12 mmol) in dry pyridine (10 mL) was added 4,4′-dimethoxytritylchloride (1.16 g, 3.41 mmol) and DMAP (10 mg) at room temperature. Thesolution was stirred for 2 hours and the product concentrated underreduced pressure. The residue was dissolved in CH₂Cl₂ (70 mL), washedwith saturated NaHCO₃ (50 mL), saturated NaCl (2×50 mL), dried withMgSO₄ and evaporated under reduced pressure. The residue was purified byshort silica gel (50 g) column chromatography. Elution withCH₂Cl₂-triethylamine (99:1), pooling and concentrating the appropriatefractions furnished the title compound (1.29 g, 90%) as a white foam.

¹H NMR (DMSO-d₆) δ 2.1 (3H, s, SCH₃), 3.5 (1H, m, 2′H), 3.75 (6H, s,OCH₃), 4.15 (1H, m, 4′ H), 4.4 (1H, t, 3′H), 5.74 (1H, br d, 3′OH), 6.15(1H, d, C1H), 6.8-8.0 (25H, m, ArH and C₅H), 8.24 (1H, d, C₆H), 11.3(1H, br s, NH).

M.2′-Deoxy-N⁴-Benzoyl-3-O—[(N,N-diisopropyl)-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiocytidine)

2′-Deoxy-N⁴-benzoyl-5′-(4,4′-dimethoxytrityl)-2′-methylthiocytidine(1.41 g, 2.07 mmol) was treated with diisopropylethylamine (1.4 mL, 8mmol) and N,N-diisopropyl-β-cyanoethylphosphoramide chloride (1.26 mL,5.34 mmol) in dry THF (25 mL) as described in Example 8-G above. Thecrude product was purified by short silica gel (50 g) columnchromatography using CH₂Cl₂-hexanes-triethylamine (89:10:1) as theeluent. The appropriate fractions were pooled and evaporated underreduced pressure to give the title compound (1.30 g, 71%) as a whitefoam (mixture of diastereoisomers).

¹H NMR (CDCl₃) δ 2.31 (3H, s, SCH₃), 3.45-3.7 (3H, m, 2′H and 5′CH₂),3.83 (6H, m, OCH₃), 4.27-4.35 (1H, m, 4′H), 4.6-4.8 (1H, m, 3′H), 6.35(1H, 2d, 1′H), 6.82-7.8 (25H, m, ArH and C₅H), 8.38 and 8.45 (1H, 2d,C₆H) and other protons.

³¹P NMR δ 151.03 and 151.08 ppm.

N. 2′-Deoxy-2′-methylsulfinylcytidine

2′-Deoxy-2′-methylthiocytidine of Example 8-J was treated as per theprocedure of Example 8-D to yield the title compound as a mixture ofdiastereoisomers having a complex ¹H NMR spectrum.

O. 2′-Deoxy-2′-methylsulfonylcytidine

2′-Deoxy-2′-methylthiocytidine of Example 8-J was treated as per theprocedure of Example 8-E to yield the title compound.

P.N⁶-Benzoyl-3′,5′-di-O-[Tetrahydropyran-2-yl]-2′-deoxy-2′-methylthioadenosine

N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adeninefrom Example 1-D is prepared by treatment with methanethiol in thepresence of tetramethylguanidine to yield the title compound.

Q. N⁶-Benzoyl-2′-deoxy-2′-methylthioadenosine

N⁶-Benzoyl-3′,5′-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenosinefrom Example 8-P is treated as per Example 1-F to yield the titlecompound.

R. N⁶-Benzoyl-2′-deoxy-2′-methylsulfinyladenosine

N⁶-Benzoyl-2′-deoxy-2′methylthioadenosine from Example 8-Q is treated asper the procedure of Example 8-D to yield the title compound.

S. N⁶-Benzoyl-2′-deoxy-2′-methylsulfonyladenosine

N⁶-Benzoyl-2′-deoxy-2′methylthioadenosine from Example 8-Q is treated asper the procedure of Example 8-E to yield the title compound.

T.N²-Isobutyryl-3′,5′-di-O-(tetrahydropyran-2-yl)-2′-deoxy-2′-methylthioguanosine

N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-arabinofuranosyl)guaninefrom Example 1-P is treated with methanethiol in the presence of1,1,3,3-tetramethylguanidine to yield the title compound.

U. N²-Isobutyryl-2′-deoxy-2′-methylthioguanosine

N²-Isobutyryl-3′,5′-di-O-(tetrahydropyran-2-yl)-2′-deoxy-2′-methylthioguanosineis treated as per the procedure of Example 1-R to yield the titlecompound.

V. N²-Isobutyryl-2′-deoxy-2′-methylsulfinyl-guanosine

N²-Isobutyryl-2′-deoxy-2′-methylthioguanosine from Example 8-U istreated as per the procedure of Example 8-D to yield the title compound.

W. N²-Isobutyryl-2′-deoxy-2′-methylsulfonyl-guanosine

N²-Isobutyryl-2′-deoxy-2′-methylthioguanosine from Example 8-U istreated as per the procedure of Example 8-E to yield the title compound.

X. 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylsulfinyluridine

2′-Deoxy-2′-methylsulfinyluridine from Example 8-D above is treated asper the procedure of Example 8-F to yield the title compound.

Y.2′-Deoxy-3′-O—[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl)-2′-methylsulfinyluridine

2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylsulfinyluridine is treatedas per the procedure of Example 8-G to yield the title compound.

Z. N⁶Benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioadenosine

N⁶-Benzoyl-2′-deoxy-2′-methylthioadenosine from Example 8-Q above istreated as per the procedure of Example 8-F to yield the title compound.

AA. N⁶Benzoyl-2′-deoxy-3′-O—[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl-2′-methylthioadenosine

N⁶-Benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioadenosineis treated as per the procedure of Example 8-G to yield the titlecompound.

BB.2′-Deoxy-N²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioadenosine

2′-Deoxy-N²-isobutyryl-2′-methylthioguanosine from Example 8-U above istreated as per the procedure of Example 8-F to yield the title compound.

CC.2′-Deoxy-N²-isobutyryl-3′-O—[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide-5′-O-(4,4′-dimethoxytrityl-2′-methylthioguanosine

2′-Deoxy-N²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioguanosineis treated as per the procedure of Example 8-G to yield the titlecompound.

DD. 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl-2′-methylsulfonyluridine

2′-Deoxy-2′-methylsulfonyluridine from Example 8-E above is treated asper the procedure of Example 8-F to yield the title compound.

EE.2′-Deoxy-3′-O—[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl-2′-methylsulfinyluridine

2′-Deoxy-5′-O-(4,4′dimethoxytrityl)-2′-methylsulfinyluridine is treatedas per the procedure of Example 8-G to yield the title compound.

Example 9 Chemical conversion of an thymine or cytosine (pyrimidine typebase) to its β-D-2′-deoxy-2′-substituted erythropentofuranosylnucleoside; 2′-substituted Ribosylation)

The thymine or cytosine type analogs are trimethylsilylated understandard conditions such as hexamethyldisilazane (HMDS) and an acidcatalyst (ie. ammonium chloride) and then treated with3,5-O-ditoluoyl-2-deoxy-2-substituted-α-D-erythropentofuranosyl chloridein the presence of Lewis acid catalysts (i.e. stannic chloride, iodine,boron tetrafluoroborate, etc.). A specific procedure has recently beendescribed by Freskos [Nucleosides & Nucleotides, 8, 1075 (1989)] inwhich copper (I) iodide is the catalyst employed.

Example 10 Chemical conversion of an adenine or guanine (purine typebase) to its β-D-2′-deoxy-2′-substituted erythropentofuranosylnucleoside; 2′-substituted Ribosylation)

The protected purine type analogs are converted to their sodium saltsvia sodium hydride in acetonitrile and are then treated with3,5-O-ditoluoyl-2-deoxy-2-substituted-α-D-erythro-pentofuranosylchloride at ambient temperature. A specific procedure has recently beendescribed by Robins et al. [Journal of American Chemical Society, 106,6379 (1984)].

Example 11 Conversion of 2′-deoxy-2-substituted thymidines to thecorresponding 2′-deoxy-2′-substituted cytidines (chemical conversion ofan pyrimidine type 4-keto group to an 4-amino Group)

The 3′ and 5′ sugar hydroxyls of the 2′modified nucleoside types areprotected by acyl groups such as toluoyl, benzoyl, p-nitrobenzoyl,acetyl, isobutryl, trifluoroacetyl, etc. under standards conditionsusing acid chlorides or anhydrides, pyridine as the solvent anddimethylaminopyridine as a catalyst. The protected nucleoside is nextchlorinated with thionyl chloride or phosphoryl chloride in pyridine oranother appropriate basic solvent. The 4-chloro group is then displacedwith ammonia in methanol. Deprotection of the sugar hydroxyls also takesplace. The amino group is benzoylated and the acyl groups areselectively removed by aqueous sodium hydroxide solution. Alternatively,the in situ process of first treating the nucleoside withchlorotrimethylsilane and base to protect the sugar hydroxyls fromsubsequent acylation may be employed. [Ogilvie, Can J. Chem., 67, 831(1989)]. Another conversion approach is to replace the 4-chloro groupwith a 1,2,4-triazolo group which remains intact throughout theoligonucleotide synthesis on the automated synthesizer and is displacedby ammonia during treatment with ammonium hydroxide which cleaves theoligonucleotide from the CPG support and effects deprotection of theheterocycle. Furthermore, in many cases the 4-chloro group can beutilized as described and replaced at the end of oligonucleotidesynthesis.

Example 12 Procedure for the attachment of 2′-deoxy-2′-substituted5′-dimethoxytriphenylmethyl ribonucleosides to the 5′-hydroxyl ofNucleosides Bound to CPG Support

The 2′-deoxy-2′-substituted nucleoside that will reside at the terminal3′-position of the oligonucleotide is protected as a 5′-DMT group (thecytosine and adenine exocyclic amino groups are benzoylated and theguanine amino is isobutrylated) and treated with trifluoroaceticacid/bromoacetic acid mixed anhydride in pyridine anddimethylaminopyridine at 50° C. for five hours. The solution is thenevaporated under reduced pressure to a thin syrup which is dissolved inethyl acetate and passed through a column of silica gel. The homogenousfractions are collected and evaporated to dryness. A solution of 10 mLof acetonitrile, 10 μM of the 3′-O-bromomethylester-modified pyrimidinenucleoside, and 1 mL of pyridine/dimethylaminopyridine (1:1) is syringedslowly (60 to 90 sec) through a 1 μM column of CPG thymidine (AppliedBiosystems, Inc.) that had previously been treated with acid accordingto standard conditions to afford the free 5′-hydroxyl group. Othernucleoside-bound CPG columns may be employed. The eluent is collectedand syringed again through the column. This process is repeated threetimes. The CPG column is washed slowly with 10 mL of acetonitrile andthen attached to an ABI 380B nucleic acid synthesizer. Oligonucleotidesynthesis is now initiated. The standard conditions of concentratedammonium hydroxide deprotection that cleaves the thymidine ester linkagefrom the CPG support also cleaves the 3′,5′ ester linkage connecting thepyrimidine modified nucleoside to the thymidine that was initially boundto the CPG nucleoside. In this manner, any 2′-substituted nucleoside orgenerally any nucleoside with modifications in the heterocycle and/orsugar can be attached at the 3′ end of an oligonucleotide.

Example 13 Procedure for the conversion of 2′-deoxy-2′-substitutedribonucleoside-5′-DMT-3′-phosphoramidites into Oligonucleotides

The polyribonucleotide solid phase synthesis procedure of Sproat et al.[Nucleic Acids Research, 17, 3373 (1989)] is utilized to prepare2′-modified oligonucleotides.

Oligonucleotides of the sequence CGACTATGCAAGTAC (SEQ ID NO:21) having2′-deoxy-2′-fluoro nucleotides were incorporated at various positionswithin this sequence. In a first oligonucleotide, each of the adenosinenucleotides at positions 3, 6, 10, 11 and 14 (5′ to 3′ direction) weremodified to include a 2′-deoxy-2′-fluoro moiety. In a furtheroligonucleotide, the adenosine and the thymidine nucleotides atpositions 3, 5, 6, 7, 10, 11, 13 and 14 were so modified. In a furtheroligonucleotide, the adenosine, thymidine and cytidine nucleotides atpositions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13 and 14 were so modified, andin even a further oligonucleotide, the nucleotides (adenosine,thymidine, cytidine and guanosine) at every position were so modified.Additionally, an oligonucleotide having the sequence CTCGTACCTTCCGGTCC(SEQ ID NO:22) was prepared having adenosine, thymidine and cytidinenucleotides at positions 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 15 and 16also modified to contain 2′-deoxy-2′-fluoro substituents.

Various oligonucleotides were prepared incorporating nucleotides having2′-deoxy-2′-methylthio substituents. For ascertaining the couplingefficiencies of 2′-deoxy-2′-methylthio bearing nucleotides intooligonucleotides, the trimer TCC and the tetramer TUU U weresynthesized. In the trimer, the central cytidine nucleotide (the secondnucleotide) included a 2′-deoxy-2′-methylthio substituent. In thetetramer, each of the uridine nucleotides included a2′-deoxy-2′methylthio substituent. In further oligonucleotides,2′-deoxy-2′-methylthio substituent bearing nucleotides were incorporatedwithin the oligonucleotide sequence in selected sequence positions. Eachof the nucleotides at the remaining sequence positions incorporated a2′-O-methyl substituent. Thus, all the nucleotides within theoligonucleotide included a substituent group thereon, either a2′-deoxy-2′-methylthio substituent or a 2′-O-methyl substituent.

These oligonucleotides are: GAGCUCCCAGGC (SEQ ID NO:23) having2′-deoxy-2′-methylthio substituents at positions 4, 5, 6, 7 and 8;CGACUAUGCAAGUAC (SEQ ID NO:24) having 2′-deoxy-2′-methylthiosubstituents at positions 1, 4, 5, 7, 9 and 13; UCCAGGUGUCCGAUC (SEQ IDNO:25) having 2′-deoxy-2′-methylthio substituents at positions 1, 2, 3,7, 9, 10, 11 and 14; TCCAGGCCGUUUC (SEQ ID NO:26) having2′-deoxy-2′-methylthio substituents at positions 10, 11 and 12; andTCCAGGTGTCCCC (SEQ ID NO:27) having 2′-deoxy-2′-methylthio substituentsat positions 10, 11 and 12.

Example 14 Preparation of 2′-Deoxy-2′-fluoro Modified PhosphorothioatesOligonucleotides

2′-Deoxy-2′-substituted 5′-DMT nucleoside 3′-phosphoramidites preparedas described in Examples 1-7 were inserted into sequence-specificoligonucleotide phosphorothioates as described by Beaucage et al.[Journal of American Chemical Society, 112, 1253 (1990)] and Sproat etal. [Nucleic Acids Research, 17, 3373 (1989)].

Oligonucleotides of the sequence CGA CTA TGC AAG TAC havingphosphorothioate backbone linkages and 2′-deoxy-2′-fluoro substituentbearing nucleotides were incorporated at various positions within thissequence. In a first oligonucleotide, each of the backbone linkages wasa phosphorothioate linkage and each of the adenosine, thymidine andcytidine nucleotides at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13 and 14(5′ to 3′ direction) were modified to include a 2′-deoxy-2′-fluoromoiety. In a further oligonucleotide, each of the backbone linkages wasa phosphorothioate linkage and the nucleotides (adenosine, thymidine,cytidine and guanosine) at every position were modified to include a2′-deoxy-2′-fluoro moiety.

Example 15 Preparation of 2′-Deoxy-2′-fluoro Modified PhosphateMethylated Oligonucleotides

The protection, tosyl chloride mediated methanolysis, and milddeprotection described by Koole et al. [Journal of Organic Chemistry,54, 1657 (1989)] is applied to 2′-substituted oligonucleotides to affordphosphate-methylated 2′-substituted oligonucleotides.

Example 16 Hybridization Analysis A. Evaluation of the Thermodynamics ofHybridization of 2′-Modified Oligonucleotides

The ability of the 2′-modified oligonucleotides to hybridize to theircomplementary RNA or DNA sequences was determined by thermal meltinganalysis. The RNA complement was synthesized from T7 RNA polymerase anda template-promoter of DNA synthesized with an Applied Biosystems, Inc.380B RNA species was purified by ion exchange using FPLC (LKB Pharmacia,Inc.). Natural antisense oligonucleotides or those containing2′-modifications at specific locations were added to either the RNA orDNA complement at stoichiometric concentrations and the absorbance (260nm) hyperchromicity upon duplex to random coil transition was monitoredusing a Gilford Response II spectrophotometer. These measurements wereperformed in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, andNaCl to yield an ionic strength of 10 either 0.1 M or 1.0 M. Data wasanalyzed by a graphic representation of 1/T_(m) vs ln [Ct], where [Ct]was the total oligonucleotide concentration. From this analysis thethermodynamic para-meters were determined. Based upon the informationgained concerning the stability of the duplex of heteroduplex formed,the placement of modified pyrimidine into oligonucleotides were assessedfor their effects on helix stability. Modifications that drasticallyalter the stability of the hybrid exhibit reductions in the free energy(delta G) and decisions concerning their usefulness as antisenseoligonucleotides were made.

As is shown in the following table (Table 1), the incorporation of2′-deoxy-2′-fluoro nucleotides into oligonucleotides resulted insignificant increases in the duplex stability of the modifiedoligonucletide strand (the antisense strand) and its complementary RNAstrand (the sense strand). In both, phosphodiester backbone andphosphorothioate backbone oligonucleotides, the stability of the duplexincreased as the number of 2′-deoxy-2′-fluoro-containing nucleotides inthe antisense strand increased. As is evident from Table 1, withoutexception, the addition of a 2′-deoxy-2′-fluoro bearing nucleotide,irrespective of the individual substituent bearing nucleotide or theposition of that nucleotide in the oligonucleotide sequence, resulted inan increase in the duplex stability.

In Table 1, the underlined nucleotides represent nucleotides thatinclude 1 2′-deoxy-2′-fluoro substituent. The oligonucleotides prefacedwith the designation “ps” have a phosphorothioate backbone. Unlabeledoligonucleotides have phosphodiester backbones.

TABLE 1 EFFECTS OF 2′-DEOXY-2′-FLUORO MODIFICATIONS ON DNA (ANTISENSE)RNA (SENSE) DUPLEX STABILITY T_(m) G° 37 G° 37 T_(m) T_(m) (° C.)/Antisense Sequence (kcal/mol) (kcal/mol) (° C.) (° C.) subst. CGA CTATGC AAG TAG −10.11 ± 0.04 45.1 (SEQ ID NO:21) CGA CTA TGC AAG TAC −13.61± 0.08  −3.50 ± 0.09 53.0 +7.9 +1.6 (SEQ ID NO:21) CGA CUA UGC AAG UAC−16.18 ± 0.08  −6.07 ± 0.09 58.9 +13.8 +1.7 (SEQ ID NO:24)CGA CUA UGC AAG UAC −19.85 ± 0.05  −9.74 ± 0.06 65.2 +20.1 +1.8 (SEQ IDNO:24) ps(CGA CTA TGC AAG TAC)  −7.58 ± 0.06 33.9 −11.2 (SEQ ID NO:21)ps(CGA CUA UGC AAG UAC) −15.90 ± 0.34  −8.32 ± 0.34 60.9 +27.0 +2.5 (SEQID NO:24) CTC GTA CCT TCC GGT CC −14.57 ± 0.13 61.6 (SEQ ID NO:22)CUC GUA CCU UCC GGU CC −27.81 ± 0.05 −13.24 ± 0.14 81.6 +1.4 (SEQ IDNO:28)

As is evident from Table 1, the duplexes formed between RNA andoligonucleotides containing 2′-deoxy-2′-fluoro substituted nucleotidesexhibited increased binding stability as measured by the hybridizationthermodynamic stability. Delta T_(m)s of greater than 20° C. weremeasured. By modifying the backbone to a phosphorothioate backbone, evengreater delta T_(m)s were observed. In this instance, delta T_(m)sgreater than 31° C. were measured. These fluoro-substitutedoligonucleotides exhibited a consistent and additive increase in thethermodynamic stability of the duplexes formed with RNA. While we do notwish to be bound by theory, it is presently believed that the presenceof a 2′-fluoro substituent results in the sugar moiety of the2′-fluoro-substituted nucleotide assuming substantially a 3′-endoconformation and this results in the oligonucleotide-RNA complexassuming an A-type helical conformation.

B. Fidelity of Hybridization of 2′-Modified Oligonucleotides

The ability of the 2′-modified antisense oligo-nucleotides to hybridizewith absolute specificity to the targeted mRNA was shown by Northernblot analysis of purified target mRNA in the presence of total cellularRNA. Target mRNA was synthesized from a vector containing the cDNA forthe target mRNA located downstream from a T7 RNA polymerase promoter.Synthesized mRNA was electrophoresed in an agarose gel and transferredto a suitable support membrane (i.e. nitrocellulose). The supportmembrane was blocked and probed using ³²P-labeled antisenseoligonucleotides. The stringency will be determined by replicate blotsand washing in either elevated temperatures or decreased ionic strengthof the wash buffer. Autoradiography was performed to assess the presenceof heteroduplex formation and the autoradiogram quantitated by laserdensitometry (LKB Pharmacia, Inc.). The specificity of hybrid formationwas determined by isolation of total cellular RNA by standard techniquesand its analysis by agarose electrophoresis, membrane transfer andprobing with the labeled 2′-modified oligonucleotides. Stringency waspredetermined for the unmodified antisense oligonucleotides and theconditions used such that only the specifically targeted mRNA wascapable of forming a heteroduplex with the 2′-modified oligonucleotide.

C. Base-Pair Specificity of Oligonucleotides and RNA

Base-pair specificity of 2′-deoxy-2′-fluoro modified oligonucleotideswith the RNA complement (a “Y” strand) was determined by effectingsingle base-pair mismatches and a bulge. The results of thesedeterminations are shown in Table 2. An 18mer “X” strand oligonucleotidecontaining 14 adenosine, thymidine and cytidine nucleotides having a2′-deoxy-2′-fluoro substituent was hybridized with the RNA complement“Y” strand in which the 10th position was varied. In Table 2, theunderlined nucleotides represent nucleotides that include a2′-deoxy-2′-fluoro substituent.

As is evident from Table 2, the 2′-deoxy-2′-fluoro modifiedoligonucleotide formed a duplex with the RNA complement with greaterspecificity than a like-sequenced unmodified oligonucleotide.

TABLE 2 EFFECTS OF SINGLE BASE MISMATCHES ON 2′-DEOXY-2′-FLUORO MODIFIEDDNA-RNA DUPLEX STABILITY X strand: deoxy (CTC GTA CCT TTC CGG TCC) (SEQID NO:29) Y strand: ribo (^(3′) GAG CAU GGY AAG GCC AGG ^(5′)) (SEQ IDNO:30) Y G° 37 G° 37 T_(m) (° C.) Base pair type (kcal/mol) (kcal/mol)(° C.) T_(m) A Watson-Crick −14.57 ± 0.13 61.6 C T-C mismatch −12.78± 0.11   1.79 ± 0.17 54.4 −7.2 G T-G mismatch −16.39 ± 0.25  −1.82± 0.28 61.7 0.1 U T-U mismatch −13.48 ± 0.17   1.09 ± 0.22 55.9 −5.7None Bulged T −14.86 ± 0.35 −0.284 ± 0.37 59.4 −2.2 X strand: deoxy (CUC  G UA   CCU   UUC   C GG  UC C) (SEQ ID NO:31) Y strand: ribo (^(3′)GAG CAU GGY AAG GCC AGG ^(5′)) (SEQ ID NO:30) Y G° 37 G° 37 T_(m) (° C.)Base pair type (kcal/mol) (kcal/mol) (° C.) T_(m) A Watson-Crick −27.80± 0.05 81.6 C U-C mismatch −21.98 ± 0.28 5.82 ± 0.28 73.8 −7.8 G U-Gmismatch −21.69 ± 0.16 6.12 ± 0.17 77.8 −3.8 U U-U mismatch −18.68± 0.15 9.13 ± 0.16 73.6 −8.0 None Bulged U −22.87 ± 0.27 4.94 ± 0.2775.5 −6.2

Example 17 Nuclease Resistance A. Evaluation of the Resistance of2′-Modified Oligonucleotides to Serum and Cytoplasmic Nucleases

Natural phosphorothioate, and 2-modified oligonucleotides were assessedfor their resistance to serum nucleases by incubation of theoligonucleotides in media containing various concentrations of fetalcalf serum or adult human serum. Labeled oligonucleotides were incubatedfor various times, treated with protease K and then analyzed by gelelectrophoresis on 20% polyacrylamide-urea denaturing gels andsubsequent autoradiography. Autoradiograms were quantitated by laserdensitometry. Based upon the location of the modifications and the knownlength of the oligonucleotide it was possible to determine the effect onnuclease degradation by the particular 2′-modification. For thecytoplasmic nucleases, a HL60 cell line was used. A post-mitochondrialsupernatant was prepared by differential centrifugation and the labeledoligonucleotides were incubated in this supernatant for various times.Following the incubation, oligonucleotides were assessed for degradationas outlined above for serum nucleolytic degradation. Autoradiographyresults were quantitated for comparison of the unmodified, thephosphorothioates, and the 2′-modified oligonucleotides.

Utilizing these test systems, the stability of a 15mer oligonucleotidehaving 2′-deoxy-2′-fluoro-substituted nucleotides at positions 12 and 14and a phosphorothioate backbone were investigated. As a control, anunsubstituted phosphodiester oligonucleotide was 50% degraded within 1hour, and 100% degraded within 20 hours. In comparison, for the2′-deoxy-2′-fluoro-substituted oligonucleotide having a phosphorothioatebackbone, degradation was limited to less that 10% after 20 hours.

B. Evaluation of the Resistance of 2′-Modified Oligonucleotides toSpecific Endo- and Exonucleases

Evaluation of the resistance of natural and 2′-modified oligonucleotidesto specific nucleases (i.e., endonucleases, 3′,5′-exo-, and5′,3′-exonucleases) was done to determine the exact effect of themodifications on degradation. Modified oligonucleotides were incubatedin defined reaction buffers specific for various selected nucleases.Following treatment of the products with protease K, urea was added andanalysis on 20% poly-acrylamide gels containing urea was done. Gelproducts were visualized by staining using Stains All (Sigma ChemicalCo.). Laser densitometry was used to quantitate the extend ofdegradation. The effects of the 2′-modifications were determined forspecific nucleases and compared with the results obtained from the serumand cytoplasmic systems.

Example 18 Oligonucleotide Synthesis

Unsubstituted and substituted oligonucleotides were synthesized on anautomated DNA synthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine. For phosphorothioateoligonucleotides, the standard oxidation bottle was replaced by 0.2 Msolution of 3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile forthe step wise thiation of the phosphite linkages. The thiation wait stepwas increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hours), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Analytical gel electrophoresis was accomplished in 20%acrylamide, 8 M urea, 454 mM Tris-borate buffer, pH=7.0.Oligonucleotides and phosphorothioates were judged, based onpolyacrylamide gel electrophoresis, to be greater than 80% full-lengthmaterial.

Example 19 Oligonucleotide Having 2′-Substituted OligonucleotidesRegions Flanking Central 2′-Deoxy Phosphorothioate OligonucleotideRegion

A 15mer RNA target of the sequence 5′GCGTTTTTTTTTTGCG 3′ (SEQ ID NO:32)was prepared in the normal manner on the DNA sequencer using RNAprotocols. A series of complementary phosphorothioate oligonucleotideshaving 2′-O-substituted nucleotides in regions that flank a 2′-deoxyregion were prepared utilizing 2′-O-substituted nucleotide precursorsprepared as per known literature preparations, i.e. 2′-O-methyl, or asper the procedure of International Publication Number WO 92/03568,published Mar. 5, 1992. The 2′-O-substituted nucleotides were added astheir 5′-O-dimethoxytrityl-3′-phosphoramidites in the normal manner onthe DNA synthesizer. The complementary oligonucleotides have thesequence of 5′ CGCAAAAAAAAAAAAACGC 3′ (SEQ ID NO:33). The2′-O-substituent was located in CGC and CG regions of theseoligonucleotides. The following 2′-O-substituents were used: 2′-fluoro;2′-O-methyl; 2′-O-propyl; 2′-O-allyl; 2′-O-aminopropoxy;2′-O-(methoxyethoxyethyl), 2′-O-imidazolebutoxy and2′-O-imidazolepropoxy.

Example 20 Ras-Luciferase Reporter Gene Assembly

The ras-luciferase reporter genes described in this study were assembledusing PCR technology. Oligonucleotide primers were synthesized for useas primers for PCR cloning of the 5′-regions of exon 1 of both themutant (codon 12) and non-mutant (wild-type) human H-ras genes. H-rasgene templates were purchased from the American Type Culture Collection(ATCC numbers 41000 and 41001) in Bethesda, Md. The oligonucleotide PCRprimers5′-ACA-TTA-TGC-TAG-CTT-TTT-GAG-TAA-ACT-TGT-GGG-GCA-GGA-GAC-CCT-GT-3′(sense) (SEQ ID NO:34), and 5′-GAG-ATC-TGA-AGC-TTC-TGG-ATG-GTC-AGC-GC-3′(antisense) (SEQ ID NO:35), were used in standard PCR reactions usingmutant and non-mutant H-ras genes as templates. These primers areexpected to produce a DNA product of 145 base pairs corresponding tosequences −53 to +65 (relative to the translational initiation site) ofnormal and mutant H-ras, flanked by NheI and HindIII restrictionendonuclease sites. The PCR product was gel purified, precipitated,washed and resuspended in water using standard procedures.

PCR primers for the cloning of the P. pyralis (firefly) luciferase genewere designed such that the PCR product would code for the full-lengthluciferase protein with the exception of the amino-terminal methionineresidue, which would be replaced with two amino acids, an amino-terminallysine residue followed by a leucine residue. The oligonucleotide PCRprimers used for the cloning of the luciferase gene were5′-GAG-ATC-TGA-AGC-TTG-AAG-ACG-CCA-AAA-ACA-TAA-AG-3′ (sense) (SEQ IDNO:36), and 5′-ACG-CAT-CTG-GCG-CGC-CGA-TAC-CGT-CGA-CCT-CGA-3′(antisense) (SEQ ID NO:37), were used in standard PCR reactions using acommercially available plasmid (pT3/T7-Luc) (Clontech), containing theluciferase reporter gene, as a template. These primers were expected toyield a product of approximately 1.9 kb corresponding to the luciferasegene, flanked by HindIII and BssHII restriction endonuclease sites. Thisfragment was gel purified, precipitated, washed and resuspended in waterusing standard procedures.

To complete the assembly of the ras-luciferase fusion reporter gene, theras and luciferase PCR products were digested with the appropriaterestriction endonucleases and cloned by three-part ligation into anexpression vector containing the steroid-inducible mouse mammary tumorvirus promotor MMTV using the restriction endonucleases NheI, HindIIIand BssHII. The resulting clone results in the insertion of H-ras 5′sequences (−53 to +65) fused in frame with the firefly luciferase gene.The resulting expression vector encodes a ras-luciferase fusion productwhich is expressed under control of the steroid-inducible MMTV promoter.

Example 21 Transfection of Cells with Plasmid DNA

Transfections were performed as described by Greenberg in CurrentProtocols in Molecular Biology, Ausubel et al., Eds., John Wiley andSons, New York, with the following modifications: HeLa cells were platedon 60 mm dishes at 5×10⁵ cells/dish. A total of 10 μg of DNA was addedto each dish, of which 9 μg was ras-luciferase reporter plasmid and 1 μgwas a vector expressing the rat glucocorticoid receptor under control ofthe constitutive Rous sarcoma virus (RSV) promoter. Calciumphosphate-DNA coprecipitates were removed after 16-20 hours by washingwith Tris-buffered saline [50 Mm Tris-Cl (pH 7.5), 150 mM NaCl]containing 3 mM EGTA. Fresh medium supplemented with 10% fetal bovineserum was then added to the cells. At this time, cells were pre-treatedwith antisense oligonucleotides prior to activation of reporter geneexpression by dexamethasone.

Example 22 Oligonucleotide Treatment of Cells

Immediately following plasmid transfection, cells were thrice washedwith OptiMEM (GIBCO), and prewarmed to 37° C. 2 ml of OptiMEM containing10 μg/ml N-[1-(2,3-diolethyloxy)propyl]-N,N,N-trimethylammonium chloride(DOTMA) (Bethesda Research Labs, Gaithersburg, Md.) was added to eachdish and oligonucleotides were added directly and incubated for 4 hoursat 37° C. OptiMEM was then removed and replaced with the appropriatecell growth medium containing oligonucleotide. At this time, reportergene expression was activated by treatment of cells with dexamethasoneto a final concentration of 0.2 μm. Cells were harvested 12-16 hoursfollowing steroid treatment.

Example 23 Luciferase Assays

Luciferase was extracted from cells by lysis with the detergent TritonX-100, as described by Greenberg in Current Protocols in MolecularBiology, Ausubel et al., Eds., John Wiley and Sons, New York. A DynatechML1000 luminometer was used to measure peak luminescence upon additionof luciferin (Sigma) to 625 μM. For each extract, luciferase assays wereperformed multiple times, using differing amounts of extract to ensurethat the data were gathered in the linear range of the assay.

Example 24 Antisense Oligonucleotide Inhibition of ras-Luciferase GeneExpression

A series of antisense phosphorothioate oligonucleotide analogs targetedto the codon-12 point mutation of activated H-ras were tested using theras-luciferase reporter gene system described in the foregoing examples.This series comprised a basic sequence and analogs of that basicsequence. The basic sequence was of known activity as reported inInternational Publication Number WO 92/22651 identified above. In boththe basic sequence and its analogs, each of the nucleotide subunitsincorporated phosphorothioate linkages to provide nuclease resistance.Each of the analogs incorporated nucleotide subunits that contained2′-O-methyl substitutions and 2′-deoxy-erythro-pentofuranosyl sugars. Inthe analogs, a subsequence of the 2′-deoxy-erythro-pentofuranosylsugar-containing subunits was flanked on both ends by subsequences of2′-O-methyl substituted subunits. The analogs differed from one anotherwith respect to the length of the subsequence of the2′-deoxy-erythro-pentofuranosyl sugar containing nucleotides. The lengthof these subsequences varied by 2 nucleotides between 1 and 9 totalnucleotides. The 2′-deoxy-erythro-pentofuranosyl nucleotidesub-sequences were centered at the point mutation of the codon-12 pointmutation of the activated ras.

The base sequences, sequence reference numbers and sequence ID numbersof these oligonucleotides (all are phosphorothioate analogs) are shownin Table 3. In this table those nucleotides identified with a “^(M)”contain a 2′-O-methyl substituent group and the remainder of thenucleotides identified with a “_(d)” are 2′-deoxy-erythro-pentofuranosylnucleotides.

TABLE 3 Chimeric 2′-O-methyl P = S oligonucleotides SEQ ID OLIGOSEQUENCE NO: 2570C_(d)C_(d)A_(d)C_(d)A_(d)C_(d)C_(d)G_(d)A_(d)C_(d)G_(d)G_(d)C_(d)G_(d)C_(d)C_(d)C_(d)1 3975C^(M)C^(M)A^(M)C^(M)A^(M)C^(M)C^(M)G^(M)A_(d)C^(M)G^(M)G^(M)C^(M)G^(M)C^(M)C^(M)C^(M)1 3979C^(M)C^(M)A^(M)C^(M)A^(M)C^(M)C^(M)G_(d)A_(d)C_(d)G^(M)G^(M)C^(M)G^(M)C^(M)C^(M)C^(M)1 3980C^(M)C^(M)A^(M)C^(M)A^(M)C^(M)C_(d)G_(d)A_(d)C_(d)GcG^(M)C^(M)G^(M)C^(M)C^(M)C^(M)1 3985C^(M)C^(M)A^(M)C^(M)A^(M)C_(d)C_(d)G_(d)A_(d)C_(d)G_(d)G_(d)C^(M)G^(M)C^(M)C^(M)C^(M)1 3984C^(M)C^(M)A^(M)C^(M)A_(d)C_(d)C_(d)G_(d)A_(d)C_(d)G_(d)G_(d)C_(d)G^(M)C^(M)C^(M)C^(M)1

FIG. 1 shows dose-response data in which cells were treated with thephosphorothioate oligonucleotides of Table 3. Oligonucleotide 2570 istargeted to the codon-12 point mutation of mutant (activated) H-ras RNA.The other nucleotides have 2′-O-methyl substituents groups thereon toincrease binding affinity with sections of various lengths ofinterspaced 2′-deoxy-erythro-pentofuranosyl nucleotides. The controloligonucleotide is a random phosphorothioate oligonucleotide analog, 20bases long. Results are expressed as percentage of luciferase activityin transfected cells not treated with oligonucleotide. As the figureshows, treatment of cells with increasing concentrations ofoligonucleotide 2570 resulted in a dose-dependent inhibition ofras-luciferase activity in cells expressing the mutant form ofras-luciferase. Oligonucleotide 2570 displays an approximate threefoldselectivity toward the mutant form of ras-luciferase as compared to thenormal form. As is further seen in FIG. 1, each of the oligonucleotides3980, 3985 and 3984 exhibited greater inhibition of ras-luciferaseactivity than did oligonucleotide 2570. The greatest inhibition wasdisplayed by oligonucleotide 3985 that has a subsequence of2′-deoxy-erythro-pentofuranosyl nucleotides seven nucleotides long.Oligonucleotide 3980, having a five nucleotide long2′-deoxy-erythro-pentofuranosyl nucleotide subsequence exhibited thenext greatest inhibition followed by oligonucleotide 3984 that has anine nucleotide 2′-deoxy-erythro-pentofuranosyl nucleotide subsequence.

FIG. 2 shows the results similar to FIG. 1 except it is in bar graphform. Further seen on FIG. 2 is the activity of oligonucleotide 3975 andoligonucleotide 3979. These oligonucleotides have subsequences of2′-deoxy-erythro-pentofuranosyl nucleotides one and three nucleotideslong, respectively. As is evident from FIG. 2, neither of theoligonucleotides having either the one or the three2′-deoxy-erythro-pentofuranosyl nucleotide subsequences showedsignificant activity. There was measurable activity for the threenucleotide subsequence oligonucleotide 3979 at the highest concentrationdose.

The increases in activity of oligonucleotides 3980, 3985 and 3984compared to oligonucleotide 2570 is attributed to the increase inbinding affinity imparted to these compounds by the 2′-O-methylsubstituent groups located on the compounds and by the RNase Hactivation imparted to these compounds by incorporation of a subsequenceof 2′-deoxy-erythro-pentofuranosyl nucleotides within the main sequenceof nucleotides. In contrast to the active compounds of the invention, itis interesting to note that sequences identical to those of the activeoligonucleotides 2570, 3980, 3985 and 3984 but having phosphodiesterlinkages in stead of the phosphorothioate linkages of the activeoligonucleotides of the invention showed no activity. This is attributedto these phosphodiester compounds being substrates for nucleases thatdegrade such phosphodiester compounds thus preventing them potentiallyactivating RNase H.

Other sugar modifications: The effects of other 2′ sugar modificationsbesides 2′-O-methyl on antisense activity in chimeric oligonucleotideshave been examined. These modifications are listed in Table 4, alongwith the T_(m) values obtained when 17mer oligonucleotides having2′-modified nucleotides flanking a 7-base deoxy gap were hybridized witha 25mer oligoribonucleotide complement as described in Example 25.

A relationship was observed for these oligonucleotides between alkyllength at the 2′ position and T_(m). As alkyl length increased, T_(m)decreased. The 2′-fluoro chimeric oligonucleotide displayed the highestT_(m) of the series.

TABLE 4 Correlation of T_(m) with Antisense Activity 2′-modified 17-merwith 7-deoxy gap CCACACCGACGGCGCCC (SEQ ID NO: 1) 2′ MODIFICATION T_(m)(° C.) IC₅₀ (nM) Deoxy 64.2 150 O-Pentyl 68.5 150 O-Propyl 70.4 70O-Methyl 74.7 20 Fluoro 76.9 10

These 2′ modified oligonucleotides were tested for antisense activityagainst H-ras using the transactivation reporter gene assay described inExample 26. All of these 2′ modified chimeric compounds inhibited rasexpression, with the 2′-fluoro 7-deoxy-gap compound being the mostactive. A 2′-fluoro chimeric oligonucleotide with a centered 5-deoxy gapwas also active.

Chimeric phosphorothioate oligonucleotides having SEQ ID NO:1 having2′-O-propyl regions surrounding a 5-base or 7-base deoxy gap werecompared to 2′-O-methyl chimeric oligonucleotides. ras expression in T24cells was inhibited by both 2′-O-methyl and 2′-O-propyl chimericoligonucleotides with a 7-deoxy gap and a uniform phosphorothioatebackbone. When the deoxy gap was decreased to five nucleotides, only the2′-O-methyl oligonucleotide inhibited ras expression.

Antisense oligonucleotide inhibition of H-ras gene expression in cancercells: Two phosphorothioate oligonucleotides (2502, 2503) complementaryto the ras AUG region were tested as described in Example 27, along withchimeric oligonucleotides (4998, 5122) having the same sequence and7-base deoxy gaps flanked by 2′-O-methyl regions. These chimericoligonucleotides are shown in Table 5.

TABLE 5 Chimeric phosphorothioate oligonucleotides having 2′-O-methylends (bold) and central deoxy gap (AUG target) SEQ ID OLIGO # DEOXYSEQUENCE NO: 2502 20 CTTATATTCCGTCATCGCTC 2 4998 7 CTTATATTCCGTCATCGCTC2 2503 20 TCCGTCATCGCTCCTCAGGG 3 5122 7 TCCGTCATCGCTCCTCAGGG 3

Compound 2503 inhibited ras expression in T24 cells by 71%, and thechimeric compound (4998) inhibited ras mRNA even further (84%inhibition). Compound 2502, also complementary to the AUG region,decreased ras RNA levels by 26% and the chimeric version of thisoligonucleotide (5122) demonstrated 15% inhibition. Also included inthis assay were two oligonucleotides targeted to the mutant codon 12.Compound 2570 (SEQ ID NO:1) decreased ras RNA by 82% and the 2′-O-methylchimeric version of this oligonucleotide with a seven-deoxy gap (3985)decreased ras RNA by 95%.

Oligonucleotides 2570 and 2503 were also tested to determine theireffects on ras expression in HeLa cells, which have a wild-type (i.e.,not activated) H-ras codon-12. While both of these oligonucleotidesinhibited ras expression in T24 cells (having activated codon-12), onlythe oligonucleotide (2503) specifically hybridizable with the ras AUGinhibited ras expression in HeLa cells. Oligonucleotide 2570 (SEQ IDNO:1), specifically hybridizable with the activated codon-12, did notinhibit ras expression in HeLa cells, because these cells lack theactivated codon-12 target.

Oligonucleotide 2570, a 17mer phosphorothioate oligonucleotidecomplementary to the codon-12 region of activated H-ras, was tested forinhibition of ras expression (as described in Example 25) in T24 cellsalong with chimeric phosphorothioate 2′-O-methyl oligonucleotides 3980,3985 and 3984, which have the same sequence as 2570 and have deoxy gapsof 5, 7 and 9 bases, respectively (shown in Table 3). The fully 2′-deoxyoligonucleotide 2570 and the three chimeric oligonucleotides decreasedras mRNA levels in T24 cells. Compounds 3985 (7-deoxy gap) and 3984(9-deoxy gap) decreased ras mRNA by 81%; compound 3980 (5-deoxy gap)decreased ras mRNA by 61%. Chimeric oligonucleotides having thissequence, but having 2′-fluoro-modified nucleotides flanking a 5-deoxy(4689) or 7-deoxy (4690) gap, inhibited ras mRNA expression in T24cells, with the 7-deoxy gap being preferred (82% inhibition, vs 63%inhibition for the 2′-fluoro chimera with a 5-deoxy gap).

Antisense oligonucleotide inhibition of proliferation of cancer cells:Three 17mer oligonucleotides having the same sequence (SEQ ID NO:1),complementary to the codon 12 region of activated ras, were tested foreffects on T24 cancer cell proliferation as described in Example 28.3985 is a full phosphorothioate oligonucleotide having a 7-deoxy gapflanked by 2′-O-methyl nucleotides, and 4690 is a full phosphorothioateoligonucleotide having a 7-deoxy gap flanked by 2′-F nucleotides(C^(F)C^(F)A^(F) C^(F)A^(F)C_(d) C_(d)G_(d)A_(d) C_(d)G_(d)G_(d)C^(F)G^(F)C^(F) C^(F)C^(F), SEQ ID NO:1, nucleotides identified with an“^(F)” contain a 2′-O-fluoro substituent group and the remainder of thenucleotides identified with a “_(d)” are 2′-deoxy-erythro-pentofuranosylnucleotides). Effects of these oligonucleotides on cancer cellproliferation correlated well with their effects on ras mRNA expressionshown by Northern blot analysis: oligonucleotide 2570 inhibited cellproliferation by 61%, the 2′-O-methyl chimeric oligonucleotide 3985inhibited cell proliferation by 82%, and the 2′-fluoro chimeric analoginhibited cell proliferation by 93%.

In dose-response studies of these oligonucleotides on cellproliferation, the inhibition was shown to be dose-dependent in the 25nM-100 nM range. IC₅₀ values of 44 nM, 61 nM and 98 nM could be assignedto oligonucleotides 4690, 3985 and 2570, respectively. The randomoligonucleotide control had no effect at the doses tested.

The effect of ISIS 2570 on cell proliferation was cell type-specific.The inhibition of T24 cell proliferation by this oligonucleotide wasfour times as severe as the inhibition of HeLa cells by the sameoligonucleotide (100 nM oligonucleotide concentration). ISIS 2570 istargeted to the activated (mutant) ras codon-12, which is present in T24but lacking in HeLa cells, which have the wild-type codon-12.

Chimeric backbone-modified oligonucleotides: Oligonucleotides discussedin previous examples have had uniform phosphorothioate backbones. The2′modified chimeric oligonucleotides discussed above are not active inuniform phosphodiester backbones. A chimeric oligonucleotide wassynthesized (ISIS 4226) having 2′-O-methyl regions flanking a5-nucleotide deoxy gap, with the gap region having a P═S backbone andthe flanking regions having a P═O backbone. Another chimericoligonucleotide (ISIS 4223) having a P═O backbone in the gap and P═S inflanking regions was also made. These oligonucleotides are shown inTable 6.

Additional oligonucleotides were synthesized, completely 2′deoxy andhaving phosphorothioate backbones containing either a singlephosphodiester (ISIS 4248), two phosphodiesters (ISIS 4546), threephosphodiesters (ISIS 4551), four phosphodiesters (ISIS 4593), fivephosphodiesters (ISIS 4606) or ten phosphodiester linkages (ISIS-4241)in the center of the molecule. These oligonucleotides are also shown inTable 6.

TABLE 6 Chimeric backbone (P = S/P = O) oligonucleo- tides having2′-O-methyl wings (bold) and central deoxy gap (backbone linkagesindicated by s (P = S) or o (P = O) SEQ # ID OLIGO P = S SEQUENCE NO:2570 16 CsCsAsCsAsCsCsGsAsCsGsGsCsGsCsCsC 1 4226 5CoCoAoCoAoCsCsGsAsCsGoGoCoGoCoCoC 1 4233 11CsCsAsCsAsCoCoGoAoCoGsGsCsGsCsCsC 1 4248 15CsCsAsCsAsCsCsGsAoCsGsGsCsGsCsCsC 1 4546 14CsCsAsCsAsCsCsGoAoCsGsGsCsGsCsCsC 1 4551 13CsCsAsCsAsCsCsGoAoCoGsGsCsGsCsCsC 1 4593 12CsCsAsCsAsCsCoGoAoCoGsGsCsGsCsCsC 1 4606 11CsCsAsCsAsCsCoGoAoCoGoGsCsGsCsCsC 1 4241 6CsCsAsCoAoCoCoGoAoCoGoGoCoGsCsCsC 1

Oligonucleotides were incubated in crude HeLa cellular extracts at 37°C. to determine their sensitivity to nuclease degradation as describedin Dignam et al. [Nucleic Acids Res., 11, 1475 (1983)]. Theoligonucleotide (4233) with a 5-diester gap betweenphosphorothioate/2′-O-methyl wings had a T_(1/2) of 7 hr. Theoligonucleotide with a five-phosphorothioate gap in aphosphorothioate/2′-O-methyl molecule had a T_(1/2) of 30 hours. In theset of oligonucleotides having one to ten diester linkages, theoligonucleotide (4248) with a single phosphodiester linkage was asstable to nucleases as was the full-phosphorothioate molecule, ISIS2570, showing no degradation after 5 hours in HeLa cell extract.Oligonucleotides with two-, three and four-diester gaps had T_(1/2) ofapproximately 5.5 hours, 3.75 hours, and 3.2 hours, and oligonucleotideswith five or ten deoxy linkages had T_(1/2) of 1.75 hours and 0.9 hours,respectively.

Antisense activity of chimeric backbone-modified oligonucleotides: Auniform phosphorothioate backbone is not required for antisenseactivity. ISIS 4226 and ISIS 4233 were tested in the ras-luciferasereporter system for effect on ras expression along with ISIS 2570 (fullyphosphorothioate/all deoxy), ISIS 3980 (fully phosphorothioate,2′-O-methyl wings with deoxy gap) and ISIS 3961 (fully phosphodiester,2′-O-methyl wings with deoxy gap). All of the oligonucleotides having aP═S (i.e., nuclease-resistant) gap region inhibited ras expression. Thetwo completely 2′deoxy oligonucleotides having phosphorothioatebackbones containing either a single phosphodiester (ISIS 4248) or tenphosphodiester linkages (ISIS 4241) in the center of the molecule werealso assayed for activity. The compound containing a single P═O was justas active as a full P═S molecule, while the same compound containing tenP═O was completely inactive.

Chimeric phosphorothioate oligonucleotides of SEQ ID NO:1 were made,having a phosphorothioate backbone in the 7-base deoxy gap region only,and phosphodiester in the flanking regions, which were either2′-O-methyl or 2′-O-propyl. The oligonucleotide with the 2′-O-propyldiester flanking regions was able to inhibit ras expression.

Example 25 Melting Curves

Absorbance vs. temperature curves were measured at 260 nm using aGilford 260 spectrophotometer interfaced to an IBM PC computer and aGilford Response II spectrophotometer. The buffer contained 100 mM Na⁺,10 mM phosphate and 0.1 mM EDTA, pH 7. Oligonucleotide concentration was4 μM each strand determined from the absorbance at 85° C. and extinctioncoefficients calculated according to Puglisi and Tinoco [Methods inEnzymol., 180, 304 (1989). T_(m) values, free energies of duplexformation and association constants were obtained from fits of data to atwo state model with linear sloping baselines. [Petersheim and Turner,Biochemistry, 22, 256 (1983). Reported parameters are averages of atleast three experiments. For some oligonucleotides, free energies ofduplex formation were also obtained from plots of T_(m) ⁻¹ vs log₁₀(concentration). Borer et al., J. Mol. Biol., 86, 843 (1974).

Example 26 ras Transactivation Reporter Gene System

The expression plasmid pSV2-oli, containing an activated (codon 12,GGC-GTC) H-ras cDNA insert under control of the constitutive SV40promoter, was a gift from Dr. Bruno Tocque (Rhone-Poulenc Sante, Vitry,France). This plasmid was used as a template to construct, by PCR, aH-ras expression plasmid under regulation of the steroid-inducible mousemammary tumor virus (MMTV) promoter. To obtain H-ras coding sequences,the 570 bp coding region of the H-ras gene was amplified by PCR. The PCRprimers were designed with unique restriction endonuclease sites intheir 5′-regions to facilitate cloning. The PCR product containing thecoding region of the H-ras codon 12 mutant oncogene was gel purified,digested, and gel purified once again prior to cloning. Thisconstruction was completed by cloning the insert into the expressionplasmid pMAMneo (Clontech Laboratories, CA).

The ras-responsive reporter gene pRDO53 was used to detect rasexpression. [Owen et al., Proc. Natl. Acad. Sci. U.S.A., 87, 3866(1990).

Example 27 Northern Blot Analysis of ras Expression In Vivo

The human urinary bladder cancer cell line T24 was obtained from theAmerican Type Culture Collection (Rockville Md.). Cells were grown inMcCoy's 5A medium with L-glutamine (GIBCO-BRL, Gaithersburg, Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of OptiMEM (GIBCO)reduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide was thenadded to the desired concentration. After 4 hours of treatment, themedium was replaced with McCoy's medium. Cells were harvested 48 hoursafter oligonucleotide treatment and RNA was isolated using a standardCsCl purification method. [Kingston in Current Protocols in MolecularBiology, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A.Smith, J. G. Seidman and K. Strahl, Eds., John Wiley and Sons, NewYork.] The human epithelioid carcinoma cell line HeLa 229 was obtainedfrom the American Type Culture Collection (Bethesda, Md.). HeLa cellswere maintained as monolayers on 6-well plates in Dulbecco's ModifiedEagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100U/ml penicillin. Treatment with oligonucleotide and isolation of RNAwere essentially as described above for T24 cells.

Northern hybridization: 10 μg of each RNA was electrophoresed on a 1.2%agarose/formaldehyde gel and transferred overnight to GeneBind 45 nylonmembrane (Pharmacia LKB, Piscataway, N.J.) using standard methods.[Kingston in Current Protocols in Molecular Biology, F. M. Ausubel, R.Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K.Strahl, Eds., John Wiley and Sons, New York.] RNA was UV-crosslinked tothe membrane. Double-stranded ³²P-labeled probes were synthesized usingthe Prime a Gene labeling kit (Promega, Madison Wis.). The ras probe wasa SalI-NheI fragment of a cDNA clone of the activated (mutant) H-rasmRNA having a GGC-to-GTC mutation at codon-12. The control probe wasG3PDH. Blots were prehybridized for 15 minutes at 68° C. with theQuickHyb hybridization solution (Stratagene, La Jolla, Calif.). Theheat-denatured radioactive probe (2.5×10⁶ counts/2 ml hybridizationsolution) mixed with 100 μl of 10 mg/ml salmon sperm DNA was added andthe membrane was hybridized for 1 hour at 68° C. The blots were washedtwice for 15 minutes at room temperature in 2×SSC/0.1% SDS and once for30 minutes at 60° C. with 0.1×SSC/0.1% SDS. Blots were autoradiographedand the intensity of signal was quantitated using an ImageQuantPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). Northern blotswere first hybridized with the ras probe, then stripped by boiling for15 minutes in 0.1×SSC/0.1% SDS and rehybridized with the control G3PDHprobe to check for correct sample loading.

Example 28 Antisense Oligonucleotide Inhibition of Proliferation ofCancer Cells

Cells were cultured and treated with oligonucleotide essentially asdescribed in Example 27. Cells were seeded on 60 mm plates and weretreated with oligonucleotide in the presence of DOTMA when they reached70% confluency. Time course experiment: On day 1, cells were treatedwith a single dose of oligonucleotide at a final concentration of 100nM. The growth medium was changed once on day 3 and cells were countedevery day for 5 days, using a counting chamber. Dose-responseexperiment: Various concentrations of oligonucleotide (10, 25, 50, 100or 250 nM) were added to the cells and cells were harvested and counted3 days later. Oligonucleotides 2570, 3985 and 4690 were tested foreffects on T24 cancer cell proliferation.

Example 29 Inhibition of PKC-α mRNA Expression by Chimeric (deoxygapped) 2′-O-methyl Oligonucleotides

Oligonucleotides having SEQ ID NO:4 were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having a centered deoxy gapof varying lengths flanked by 2′-O-methylated regions. Theseoligonucleotides (500 nM concentration) were tested for effects on PKC-αmRNA levels by Northern blot analysis. Deoxy gaps of eight nucleotidesor more gave maximal reduction of PKC-α mRNA levels (both transcripts)in all cases. These oligonucleotides reduced PKC-α mRNA by approximately83% with a deoxy gap length of four nucleotides, and gave nearlycomplete reduction of PKC-α mRNA with a deoxy gap length of six or more.

The 2′-O-methyl chimeric oligonucleotides with four- or six-nucleotidedeoxy gaps have an IC₅₀ for PKC-α mRNA reduction (concentration ofoligonucleotide needed to give a 50% reduction in PKC-α mRNA levels) of200-250 nM, as did the full-deoxy oligonucleotide (all arephosphorothioates throughout). The 2′-O-methyl chimeric oligonucleotidewith an 8-nucleotide deoxy gap had an IC₅₀ of approximately 85 nM.

Several variations of this chimeric oligonucleotide (SEQ ID NO:4) werecompared for ability to lower PKC-α mRNA levels. These oligonucleotidesare shown in Table 7.

TABLE 7 Chimeric 2′-O-methyl/deoxy P = S oligonucleo- tides bold= 2′-O-methyl; s = P = S linkage, o = P = O linkage SEQ ID OLIGOSEQUENCE NO: 3522 AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 4 5352AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 4 6996AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC 4 7008AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 4 7024AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC 4

Effect of these oligonucleotides on PKC-α mRNA levels is shown in FIG.3. Oligonucleotides 7008, 3522 and 5352 show reduction of PKC-α mRNA,with 5352 being most active.

A series of 2′-O-propyl chimeric oligonucleotides was synthesized havingSEQ ID NO:4. These oligonucleotides are shown in Table 8.

TABLE 8 Chimeric 2′-O-propyl/deoxy P = S oligonucleo- tides bold= 2′-O-propyl; s = P = S linkage, o = P = O linkage SEQ ID OLIGOSEQUENCE NO. 7199 AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 4 7273AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC 4 7294AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 4 7295AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC 4

These 2′-O-propyl chimeric oligonucleotides were compared to the2′-O-methyl chimeric oligonucleotides. Oligonucleotides 7273 and 7294were more active than their 2′-O-methyl counterparts at lowering PKC-αmRNA levels. This is shown in FIGS. 4 and 5.

Example 30 Additional Oligonucleotides which Decrease PKC-α mRNAExpression

Additional phosphorothioate oligonucleotides targeted to the human PKC-α3′ untranslated region were designed and synthesized. These sequencesare shown in Table 9.

TABLE 9 Chimeric 2′-O-propyl/deoxy P = S oligonucleo- tides targeted toPKC-α 3′-UTR bold = 2′-O- propyl; s = P = S linkage, o = P = O linkageSEQ ID OLIGO SEQUENCE NO: 6632 TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC5 6653 TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC 5 6665 ToToCo TsCsGsCsTsGs GsTsGs AsGsTo ToToC 5 7082 TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC6 7083 TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC 6 7084 ToCoTo CsGsCsTsGsGs TsGsAs GsToTo ToC 6

Oligonucleotides 6632, 6653, 7082 and 7083 are most active in reducingPKC-α mRNA levels.

Example 31 Inhibition of c-raf Expression by Chimeric Oligonucleotides

Chimeric oligonucleotides having SEQ ID NO:7 were designed using theGenbank c-raf sequence HUMRAFR (Genbank listing x03484), synthesized andtested for inhibition of c-raf mRNA expression in T24 bladder carcinomacells using a Northern blot assay. These chimeric oligonucleotides havecentral “gap” regions of 6, 8 or 10 deoxynucleotides flanked by tworegions of 2′-O-methyl modified nucleotides, and are shown in Table 10.Backbones were uniformly phosphorothioate. In a Northern blot analysis,as described in Example 32, all three of these oligonucleotides (ISIS6720, 6-deoxy gap; ISIS 6717, 8-deoxy gap; ISIS 6729, 10-deoxy gap)showed greater than 70% inhibition of c-raf mRNA expression in T24cells. These oligonucleotides are preferred. The 8-deoxy gap compound(6717) showed greater than 90% inhibition and is more preferred.

TABLE 10 Chimeric 2′-O-methyl P = S deoxy “gap” oligo- nucleotides bold= 2′-O-methyl SEQ Target ID OLIGO SEQUENCE site NO: 6720TCCCGCCTGTGACATGCATT 3′UTR 7 6717 TCCCGCCTGTGACATGCATT 3′UTR 7 6729TCCCGCCTGTGACATGCATT 3′UTR 7

Additional chimeric oligonucleotides were synthesized having one or moreregions of 2′-O-methyl modification and uniform phosphorothioatebackbones. These are shown in Table 11. All are phosphorothioates; boldregions indicate 2′-O-methyl modified regions.

TABLE 11 Chimeric 2′-O-methyl P = S c-raf oligonucleotides SEQ Target IDOLIGO SEQUENCE site NO: 7848 TCCTCCTCCCCGCGGCGGGT 5′UTR 8 7852TCCTCCTCCCCGCGGCGGGT 5′UTR 8 7849 CTCGCCCGCTCCTCCTCCCC 5′UTR 9 7851CTCGCCCGCTCCTCCTCCCC 5′UTR 9 7856 TTCTCGCCCGCTCCTCCTCC 5′UTR 10 7855TTCTCGCCCGCTCCTCCTCC 5′UTR 10 7854 TTCTCCTCCTCCCCTGGCAG 3′UTR 11 7847CTGGCTTCTCCTCCTCCCCT 3′UTR 12 7850 CTGGCTTCTCCTCCTCCCCT 3′UTR 12 7853CCTGCTGGCTTCTCCTCCTC 3′UTR 13

When tested for their ability to inhibit c-raf mRNA by Northern blotanalysis, ISIS 7848, 7849, 7851, 7856, 7855, 7854, 7847, and 7853 gavebetter than 70% inhibition and are therefore preferred. Of these, 7851,7855, 7847 and 7853 gave greater than 90% inhibition and are morepreferred.

Additional chimeric oligonucleotides with various 2′ modifications wereprepared and tested. These are shown in Table 12. All arephosphorothioates; bold regions indicate 2′-modified regions.

TABLE 12 Chimeric 2′-modified P = S c-raf oligonucleotides OLIGO TARGETSEQ NO: SEQUENCE SITE MODIFIC. ID 6720 TCCCGCCTGTGACATGCATT 3′UTR2′-O-Me 7 6717 TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Me 7 6729TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Me 7 8097 TCTGGCGCTGCACCACTCTC 3′UTR2′-O-Me 14 9270 TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Pr 7 9058TCCCGCCTGTGACATGCATT 3′UTR 2′-F 7 9057 TCTGGCGCTGCACCACTCTC 3′UTR 2′-F14

Of these, oligonucleotides 6720, 6717, 6729, 9720 and 9058 arepreferred. Oligonucleotides 6717, 6729, 9720 and 9058 are morepreferred.

Example 32 Northern Blot Analysis of Inhibition of c-raf mRNA Expression

The human urinary bladder cancer cell line T24 was obtained from theAmerican Type Culture Collection (Rockville, Md.). Cells were grown inMcCoy's 5A medium with L-glutamine (GIBCO-BRL, Gaithersburg, Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of OptiMEMreduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide withlipofectin was then added to the desired concentration. After 4 hours oftreatment, the medium was replaced with McCoy's medium. Cells wereharvested 24 to 72 hours after oligonucleotide treatment and RNA wasisolated using a standard CsCl purification method. [Kingston in CurrentProtocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston,D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, Eds., John Wileyand Sons, New York.] Total RNA was isolated by centrifugation of celllysates over a CsCl cushion. RNA samples were electrophoresed through1.2% agarose-formaldehyde gels and transferred to hybridizationmembranes by capillary diffusion over a 12-14 hour period. The RNA wascross-linked to the membrane by exposure to UV light in a Stratalinker(Stratagene, La Jolla, Calif.) and hybridized to random-primed³²P-labeled c-raf cDNA probe (obtained from ATCC) or G3PDH probe as acontrol. RNA was quantitated using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

Example 33 Oligonucleotide Inhibition of Rev Gene Expression

The chimeric oligonucleotides used in this assay are shown in Table 13below.

TABLE 13 Chimeric 2′-O-propyl/deoxy P = S oligonucleo- tides targeted toHIV rev gene bold = 2′O- propyl; s = P = S linkage; o = P = O linkageSEQ ID OLIGO SEQUENCE NO: 8907 UoAoGoGoAoGoAsUsGsCsCsUsAsAoGoGoCoUoUoU15 8908 GoCoUoAoUoGoUsCsGsAsCsAsCsCoCoAoAoUoUoC 16 8909CoAoUoAoGoGoAsGsAsUsGsCsCsUoAoAoGoGoCoT 17

Transfection and Luciferase assay: 3T3 cells were maintained in DMEMwith glucose, L-glutamine, sodium pyruvate and 10% fetal bovine serum(GIBCO). For all experiments, cells were seeded the previous night at75,000 cells/well in 6-well plates (Falcon). Transfections wereperformed using the standard CaPO₄ method. For each set of replicates,15 μg/mL of pSG5/rev plasmid, 18 μg/mL pHIVenu-luc and 2 μg/mL of Rep 6were precipitated and 200 μL of this was dripped on each well. Theprecipitate was allowed to incubate on cells for 7 hours at 37° C. Themedia was then aspirated, the cells washed once with PBS, and freshcomplete media added for overnight incubation. Following incubation, themedia was removed, cells washed with 2 mL of OPTIMEM (GIBCO) and 1 mL ofOPTIMEM containing 2.5 μg/mL of Lipofectin (GIBCO-BRL) and theoligonucleotide added. The mixture was incubated for 4 hours at 37° C.,at which point it was aspirated off the cells and complete media wasadded. Two hours after this treatment, 0.2 μM/mL of dexamethasone(Sigma) was added to all wells to allow induction of the MMTV promoterof pHIVenu-luc.

The Luciferase assay was performed 24 hours later, as follows: The wellswere washed twice with PBS and the cells were harvested by scraping in200 μL of lysis buffer (1% Triton, 25 mM glycylglycine, pH 7.8, 15 mMMgSO₄, 4 mM EGTA and 1 mM DTT)> The lysate was clarified by microfugingfor 5 minutes at 11,500 rpm in the cold. 100 μL of the lysate was thencombined in a microtiter plate with 50 μL of assay buffer (25 mMglycylglycine, pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 15 mM potassiumphosphate, pH 7.8, 1 mM DTT and 7.5 mM ATP). Luc detection was performedusing a microtiter luminescent reader (Dynatech Laboratories). Thereactions were started by injecting 50 μL of 1× luciferase solution(Sigma). The 1× solution was diluted in luciferin buffer (25 mMglycylglycine, pH 7.8, 15 mM MgSO₄, 4 mM EGTA and 4 mM DTT) prior to usefrom a 10× stock (10 mM luciferin in 10 mM DTT). Samples were countedfor 20 seconds. The kinetics of firefly luc light emission arecharacterized by a flash period lasting a few seconds followed by aperiod of lower light intensity emission lasting several minutes.

Rev and RRE RNA synthesis: pSG %-Rev contains the Rev gene adjacent to aT7 promoter. Bg1II linearized pSG5-Rev was used as a DNA template fortranscription with T7 RNA polymerase. A template for the production ofRRE RNA was produced by PCR. For RNA synthesis, DNA templates were usedat 0.2 to 1.0 mg/mL, with 5 mM each of ATP, CTP and GTP, 0.5 mM of UTP,10 mM of DTT, 40 mM of Tris-HCl, pH 7.5, 6 mM of MgCl₂, 4 mM ofSpermidine, 500 U/mL of RNAsin at 20 U/μL, 2500 μCi/mL of α ³²P UTP at10 mCi/mL and 1000 U/mL of T7 RNA polymerase. The reaction was incubatedfor 1 hour at 37° C. The transcription reaction was terminated by addingformamide loading buffer and was run in a denaturing polyacrylamide gelcontaining 8 M urea. The RNA was eluted from the gel according to theprocedure of Schwartz et al. (Gene, 1990, 88, 197).

Example 34 Immunoassay for Antiviral Screening

NHDF cells were seeded in 96-well culture plates at a density of 15,000cells/well in serum-free FGM. Established monolayers were pretreatedwith the oligonucleotide overnight in FGM prior to infection. Afterpretreatment, cells were rinsed thrice with fresh, prewarmed FGM, andvirus in 100 μL of FGM/well was added to achieve an MOI of 0.05PFU/cell. After 2 hours of incubation at 37° C., virus was removed andfresh medium (100 μL/well) containing the oligonucleotide was added.Medium was exchanged 2 days after infection with fresh medium containingthe oligonucleotide, and 6 days after infection, the cells were fixed inabsolute ethanol and dried in preparation for antibody staining. Amodified protocol was used for some assays in which FGM was supplementedwith low levels of FBS (0.2%), and the incubation period after infectionwas shortened from 6 days to 3 days. The shorter assay eliminated theneed to exchange medium 2 days after infection. Both assays yieldedcomparable values for 50% effective concentrations (EC50s).

Fixed cells were blocked in a solution of PBS containing 2% bovine serumalbumin (BSA), and mouse monoclonal antibody (1H10, supplied by EisaiCo., Ltd., Japan) was added in a 1:2000 dilution in PBS-1% BSA. The 1H10antibody recognizes an abundant late HCMV polypeptide approximately 65kDa in size. Detection of bound monoclonal antibody was facilitated withbiotinylated goat anti-mouse immunoglobulin G abd streptavidin-coupledβ-galactosidase (GIBCO-BRL, Gaithersburg, Md.). Chlorophenol redβ-D-galactopyranoside was used as a substrate for β-galactosidase, andactivity was determined by measuring the optical density at 575 nm ofindividual wells with a BioTex model EL312e microplate reader.

The oligonucleotides used in this assay are shown in Table 14 below.

TABLE 14 Inhibition of CMV replication by chimeric 2′-O-methyl P = Soligonucleotides bold = 2′-O-methyl SEQ ID OLIGO SEQUENCE NO: 4325 GCGUUT GCT CTT CTT CUU GCG 18 4326 GCG UUU GCT CTT CTU CUU GCG 19

Example 35 Diagnostic Assay for the Detection of mRNA overexpression

Oligonucleotides are radiolabeled after synthesis by ³²P labeling at the5′ end with polynucleotide kinase. Sambrook et al. [“Molecular Cloning.A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 1989, Volume2, pg. 11.31-11.32]. Radiolabeled oligonucleotide is contacted withtissue or cell samples suspected of mRNA overexpression, such as asample from a patient, under conditions in which specific hybridizationcan occur, and the sample is washed to remove unbound oligonucleotide. Asimilar control is maintained wherein the radiolabeled oligonucleotideis contacted with normal cell or tissue sample under conditions thatallow specific hybridization, and the sample is washed to remove unboundoligonucleotide. Radioactivity remaining in the sample indicates boundoligonucleotide and is quantitated using a scintillation counter orother routine means. Comparison of the radioactivity remaining in thesamples from normal and diseased cells indicates overexpression of themRNA of interest.

Radiolabeled oligonucleotides of the invention are also useful inautoradiography. Tissue sections are treated with radiolabeledoligonucleotide and washed as described above, then exposed tophotographic emulsion according to standard autoradiography procedures.A control with normal cell or tissue sample is also maintained. Theemulsion, when developed, yields an image of silver grains over theregions overexpressing the mRNA, which is quantitated. The extent ofmRNA overexpression is determined by comparison of the silver grainsobserved with normal and diseased cells.

Analogous assays for fluorescent detection of mRNA expression useoligonucleotides of the invention which are labeled with fluorescein orother fluorescent tags. Labeled DNA oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidites arepurchased from Glen Research (Sterling, Va.). Incubation ofoligonucleotide and biological sample is carried out as described forradiolabeled oligonucleotides except that instead of a scintillationcounter, a fluorescence microscope is used to detect the fluorescence.Comparison of the fluorescence observed in samples from normal anddiseased cells enables detection of mRNA overexpression.

Example 36 Detection of Abnormal mRNA Expression

Tissue or cell samples suspected of expressing abnormal mRNA areincubated with a first ³²P or fluorescein-labeled oligonucleotide whichis targeted to the wild-type (normal) mRNA. An identical sample of cellsor tissues is incubated with a second labeled oligonucleotide which istargeted to the abnormal mRNA, under conditions in which specifichybridization can occur, and the sample is washed to remove unboundoligonucleotide. Label remaining in the sample indicates boundoligonucleotide and can be quantitated using a scintillation counter,fluorimeter, or other routine means. The presence of abnormal mRNA isindicated if binding is observed in the case of the second but not thefirst sample.

Double labeling can also be used with the oligonucleotides and methodsof the invention to specifically detect expression of abnormal mRNA. Asingle tissue sample is incubated with a first ³²P-labeledoligonucleotide which is targeted to wild-type mRNA, and a secondfluorescein-labeled oligonucleotide which is targeted to the abnormalmRNA, under conditions in which specific hybridization can occur. Thesample is washed to remove unbound oligonucleotide and the labels aredetected by scintillation counting and fluorimetry. The presence ofabnormal mRNA is indicated if the sample does not bind the ³²P-labeledoligonucleotide (i.e., is not radioactive) but does retain thefluorescent label (i.e., is fluorescent).

Example 37 Plasma Uptake and Tissue Distribution of Oligonucleotides inMice

The following oligonucleotides were prepared:

SEQ ID NO:20 UsGsCsAsTsCsCsCsCsCsAsGsGsCsCsAsCsCsAsT, SEQ ID NO:20UsGsCsAsTsCsCsCsCsAsGsGsCsCsAsCsCsAsT, SEQ ID NO:20UsGsCsAsTsCsCCCCAGGCsCsAsCsCsAsT,wherein bold type indicated a 2′-O-propyl substituent, “s” indicates aphosphorothioate linkage and the absence of “s” indicates aphosphodiester linkage in the respective oligonucleotides. The firstoligonucleotide is identified as Isis 3082, the second as Isis 9045 andthe third as Isis 9046 in the FIGS. 6, 7, 8 and 9. The oligonucleotideswere tritiated as per the procedure of Graham et al., Nuc. Acids Res.,1993, 16, 3737-3743.

Animals and Experimental Procedure

For each oligonucleotide studied, twenty male Balb/c mice (CharlesRiver), weighing about 25 gm, were randomly assigned into one of fourtreatment groups. Following a one-week acclimation, mice received asingle tail vein injection of ³H-radiolabeled oligonucleotide(approximately 750 nmoles/kg; ranging from 124-170 μC1/kg) administeredin phosphate buffered saline, pH 7.0. The concentration ofoligonucleotide in the dosing solution was approximately 60 μM. Oneretro-orbital bleed (at either 0.25, 0.5, 2, or 4 hours post-dose) and aterminal bleed (either 1, 3, 8 or 24 hours post-dose) was collected fromeach group. The terminal bleed was collected by cardiac puncturefollowing ketamine/xylazine anesthesia. An aliquot of each blood samplewas reserved for radioactivity determination and the remaining blood wastransferred to an EDTA-coated collection tube and centrifuged to obtainplasma. Urine and feces were collected at intervals (0-4, 4-8 and 8-24hours) from the group terminated at 24 hours.

At termination, the liver, kidneys, spleen, lungs, heart, brain, sampleof skeletal muscle, portion of the small intestine, sample of skin,pancreas, bone (both femurs containing marrow) and two lymph nodes werecollected from each mouse and weighed. Feces were weighed, thenhomogenized 1:1 with distilled water using a Brinkmann Polytronhomogenizer (Westbury, N.Y.). Plasma, tissues, urine and feceshomogenate were divided for the analysis of radioactivity by combustionand for determination of intact oligonucleotide content. All sampleswere immediately frozen on dry ice after collection and stored at −80°C. until analysis.

Analysis of Radioactivity in Plasma, Tissue, and Excreta

Plasma and urine samples were weighed directly into scintillation vialsand analyzed directly by liquid scintillation counting after theaddition of 15 ml of BetaBlend (ICN Biomedicals, Costa Mesa, Calif.).All other samples (tissues, blood and homogenized feces) were weighedinto combustion boats and oxidized in a Biological Materials Oxidizer(Model OX-100; R. J. Harvey Instrument Corp., Hillsdale, N.J.). The ³H₂Owas collected in 20 ml of cocktail, composed of 15 ml of BetaBlend and 5ml of Harvey Tritium Cocktail (R. J. Harvey Instrument Corp., Hillsdale,N.J.). The combustion efficiency was determined daily by combustion ofsamples spiked with a solution of ³H-mannitol and ranged between73.9-88.3%. Liquid scintillation counting was performed using a BeckmanLS 9800 or LS 6500 Liquid Scintillation System (Beckman Instruments,Fullerton, Calif.). Samples were counted for 10 minutes with automaticquench correction. Disintergration per minute values were corrected forthe efficiency of the combustion process.

Analysis of Data

Radioactivity in samples was expressed as disintergrations per minuteper gram of sample. These values were divided by the specific activityof the radiolabel to express the data in nanomole-equivalents of totaloligonucleotide per gram of sample, then converted to percent of doseadministered per organ or tissue. Assuming a tissue density of 1 gm/ml,the nmole/gram data were converted to a total μM concentration. Tocalculate the concentration of intact oligonucleotide in plasma, liveror kidney at each time point, the mean total μM concentrations weredivided by the percent of intact oligonucleotide in the dosing solution(82-97%), then multiplied by the mean percentage of intactoligonucleotide at each time point as determined by CGE or HPLC. Thisdata was then used for the calculation of tissue half-lives by linearregression and to compare the plasma pharmacokinetics of the differentmodified oligonucleotides. The pharmacokinetic parameters weredetermined using PCNONLIN 4.0 (Statistical Consultants, Inc., Apex,N.C.). After examination of the data, a one-compartment bolus input,first order output model (library model 1) was selected for use.

The result of the animal plasma uptake and tissue distribution tests areillustrated graphically in FIGS. 6, 7, 8 and 9. As is seen in FIG. 6,plasma concentration of each of the test oligonucleotides decrease fromthe initial injection levels to lower levels over the twenty-four hourtest period. Plasma concentrations of the oligonucleotides of theinvention were maintained at levels equivalent to those of thenon-conjugate bearing phosphorothioate. All of the test compounds weretaken up from the plasma to tissues as is shown in FIGS. 7, 8 and 9. Thecompounds of the invention had different distribution between thevarious tissues. FIG. 7 shows the distribution pattern for the controloligonucleotide, identified as ISIS 3082, a phosphorothioateoligonucleotide. FIG. 8 shows the distribution pattern for a firstcompound of the invention, an oligonucleotide, identified as ISIS 9045,having a 2′-substituent at each nucleotide. FIG. 9 shows thedistribution pattern for a further compound of the invention, a “gapmer” oligonucleotide, identified as ISIS 9046, having a 2′-substituentand phosphodiester linkages at each nucleotide at “flanking” sections ofthe oligonucleotide and 2′-deoxy, phosphorothioate nucleotides in acentral or gap region.

1. A compound comprising at least 12 covalently-bound nucleosides thatindividually include a ribose or deoxyribose sugar portion and a baseportion, wherein: said nucleosides are joined together byinternucleoside linkages such that the base portion of said nucleosidesform a mixed base sequence; and wherein at least five of saidnucleosides includes a modified ribofuranosyl moiety bearing a 2′-fluorosubstituent.
 2. A compound comprising at least 12 covalently-boundnucleosides that individually include a ribose or deoxyribose sugarportion and a base portion, wherein: said nucleosides are joinedtogether by internucleoside linkages such that the base portion of saidnucleosides form a mixed base sequence; and wherein at least twoconsecutive nucleosides in said mixed base sequence are nucleosides thatinclude a modified ribofuranosyl moiety bearing a 2′-fluoro substituent.3. A compound of claim 2 wherein at least three consecutive nucleosidesin said mixed base sequence are nucleosides that include a modifiedribofuranosyl moiety bearing a 2′-fluoro substituent.
 4. A compoundcomprising at least 12 covalently-bound nucleosides that individuallyinclude a ribose or deoxyribose sugar portion and a base portion,wherein: said nucleosides are joined together by internucleosidelinkages such that the base portion of said nucleosides form a mixedbase sequence; and wherein at least one adenosine, thymidine or cytidinenucleoside of said mixed base sequence includes a modified ribofuranosylmoiety bearing a 2′-fluoro substituent.
 5. A compound comprising atleast 12 covalently-bound nucleosides that individually include a riboseor deoxyribose sugar portion and a base portion, wherein: saidnucleosides are joined together by internucleoside linkages such thatthe base portion of said nucleosides form a mixed base sequence; andwherein at least two cytidine nucleosides consecutively located in saidmixed base sequence include a modified ribofuranosyl moiety bearing a2′-fluoro substituent.